Biophotonic compositions for the treatment of otitis externa

ABSTRACT

The present disclosure describes methods and uses of biophotonic compositions which comprise at least one oxidant and at least one chromophore capable of activating the oxidant, in association with a pharmacologically acceptable carrier for the treatment of otitis externa.

RELATED APPLICATIONS

This application claims priority to and benefit from U.S. ProvisionalPatent Application No. 62/277,263, filed Jan. 11, 2016, the disclosureof which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This description relates to the field of biophotonic compositions,methods, and uses for treating otitis externa.

BACKGROUND

Otitis externa is an inflammation or infection of the external auditorycanal, the pinna (auricle), or both. The disease can be a localcondition (e.g., atopic dermatitis, food hypersensitivity, parasiticdiseases, or seborrhea, etc.) or part of a systemic disease (e.g., foodallergy, endocrinopathies, etc.). The disease arises from primarycauses, such as foreign bodies, infections, parasites, glandularhyperplasia, etc., and secondary causes, such as bacteria, chemicals,and yeast. Usually secondary causes are complicating factors from apreviously existing condition that can lead to otitis externa if one ormore predisposing factors are present at the same time. For instance,predisposing factors are able to alter the ventilation of the externalauditory canal, thus increasing the humidity and temperature of theexternal auditory canal (e.g., obstructive diseases such as neoplasms,frequent baths, swimming, abundant hairs inside the external auditorycanal, pendulous pinna, hypertrichlosis, etc.). The presence ofpredisposing factors tends to be the cause of disease persistence, anduntreated otitis externa can lead to irreversible changes of theexternal auditory canal, such as metaplasia and ossification, and theconcomitant presence of otitis media. For these reasons, chronic otitisexterna is a difficult disease to manage and treat.

One of the main aspects involved in the chronicity of the disease is thepresence of infections caused by bacteria resistant to differentantibiotics (e.g., Pseudomonas aeruginosa). To manage such conditions,usually, both topical and systemic long lasting (1 to 2 months)antibiotic treatments are needed, followed by anti-inflammatory drugs.Furthermore, in an occurrence of purulent discharge, the externalauditory canal has to be rinsed and cleaned with specific detergents;otherwise topical antibiotics may be ineffective. Nevertheless, evenwith topical antibiotics, without solving the primary cause, thecondition will inevitably relapse. Different surgical procedures arepossible (e.g., from lateral ear canal resection to total ear canalablation with lateral osteotomy of the tympanic bulla), but theseprocedures leave a permanent aftermath.

Otitis externa is a common inflammatory disease in mammals, such ashumans, dogs, and cats. For instance, swimmers are particularly prone tootitis externa due to repeated exposure to water. Additionally, otitisexterna afflicts up to 10-20% of dogs visited in a first opinionveterinary clinic.

Effective treatments of otitis externa and chronic or relapsing otitisexterna are needed.

SUMMARY OF DISCLOSURE

In some aspects, the present disclosure provides a method of treatingotitis externa or chronic otitis externa comprising: (1) applying abiophotonic composition to a patient in need thereof, wherein thebiophotonic composition comprises at least one oxidant and at least onechromophore capable of activating the oxidant; and (2) exposing saidbiophotonic composition to actinic light for a time sufficient for saidchromophore to cause activation of said oxidant. In certain suchaspects, the patient being treated is a mammal, such as a human, afeline, or a canine. In certain embodiments, the composition is appliedto an auricle and/or ear canal of the patient.

In some embodiments, said biophotonic composition is exposed to actiniclight for a period of less than 5 minutes, e.g., for a period of fromabout 1 second to about 5 minutes. In certain embodiments, saidbiophotonic composition is exposed to actinic light for a period of lessthan about 5 minutes per cm² of an area to be treated, e.g., for aperiod of from about 1 second to about 5 minutes per cm².

In some embodiments, the source of actinic light is positioned over anarea to be treated. In certain embodiments, said actinic light isvisible light having a wavelength between about 400 nm and about 700 nm.

In some embodiments, the oxidant is chosen from hydrogen peroxide,carbamide peroxide, and benzoyl peroxide, such as carbamide peroxide. Insome embodiments, the oxidant is chosen from a peroxy acid and an alkalimetal percarbonate. In some embodiments, the oxidant is present in anamount of from about 1% to about 10% by weight of the total composition,such as from about 3% to 9%, from about 4% to 8%, from about 5% to 7%.

In some embodiments, the composition further comprises at least onehealing factor chosen from hyaluronic acid, glucosamine, and allantoin.

In some embodiments, the composition further comprises at least onegelling agent, such as glucose, modified starch, methyl cellulose,carboxymethyl cellulose, propyl cellulose, hydroxypropyl cellulose, acarbomer, alginic acid, sodium alginate, potassium alginate, ammoniumalginate, calcium alginate, agar, carrageenan, locust bean gum, pectin,or gelatin.

In some embodiments, the chromophore is chosen from a xanthenederivative dye, an azo dye, a biological stain, and a carotenoid. Incertain such embodiments, said xanthene derivative dye is chosen from afluorene dye (e.g., a pyronine dye, such as pyronine Y or pyronine B, ora rhodamine dye, such as rhodamine B, rhodamine G, or rhodamine WT), afluorone dye (e.g., fluorescein or fluorescein derivatives, such asphloxine B, rose bengal, merbromine, Eosin Y, Eosin B, or Erythrosine B,i.e., Eosin Y), or a rhodole dye. In certain such embodiments, said azodye is chosen from methyl violet, neutral red, para red, amaranth,carmoisine, allura red AC, tartrazine, orange G, ponceau 4R, methyl red,and murexide-ammonium purpurate. In certain such embodiments, saidbiological stain is chosen from saffranin O, basic fuchsin, acidfuschin, 3,3′ dihexylocarbocyanine iodide, carminic acid, andindocyanine green. In certain such embodiments, said carotenoid ischosen from crocetin, a-crocin (S,S-diapo-S,S-carotenoic acid),zeaxanthine, lycopene, α-carotene, β-carotene, bixin, and fucoxanthine.In certain such embodiments, said carotenoid is present in thecomposition as a mixture chosen from saffron red powder, annattoextract, and brown algae extract.

In some embodiments, said composition further comprises at least onechelating agent chosen from ethylenediaminetetraacetic acid (EDTA) andethylene glycol tetraacetic acid (EGTA).

In some embodiments, the patient is treated once for one or more weeks,such as once for one week, once for two weeks, once for three weeks,once for four weeks, once for five weeks, once for six weeks. In someembodiments, the patient is treated twice for one or more weeks, such astwice for one week, twice for two weeks, twice for three weeks, twicefor four weeks, twice for five weeks, twice for six weeks, twice forseven weeks.

In some aspects, the present disclosure provides for use of abiophotonic composition for the manufacture of a medicament for treatinga patient afflicted with otitis externa or chronic otitis externa,wherein said composition comprises: at least one oxidant, and at leastone chromophore capable of activating the oxidant; in association with asuitable carrier, such as a pharmacologically acceptable carrier. Incertain such aspects, the patient is a mammal, such as a human, a felineor a canine.

In some aspects, the present disclosure provides for use of abiophotonic composition for the treatment of a patient afflicted withotitis externa or chronic externa, wherein said composition comprises:at least one oxidant, and at least one chromophore capable of activatingthe oxidant; in association with a suitable carrier, such as apharmacologically acceptable carrier. In certain such aspects, thepatient is a mammal, such as a human, a feline or a canine.

Definitions

Before continuing to describe the present disclosure in further detail,it is to be understood that this disclosure is not limited to specificcompositions or process steps, as such may vary. It must be noted that,as used in this disclosure and the appended embodiments, the singularform “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise.

It is understood that, whether the term “about” is used explicitly ornot, every quantity given herein is meant to refer to the actual givenvalue, and it is also meant to refer to the approximation to such givenvalue that would reasonably be inferred based on the ordinary skill inthe art, including equivalents and approximations due to theexperimental and/or measurement conditions for such given value.

It is convenient to point out here that “and/or” where used herein is tobe taken as specific disclosure of each of the two specified features orcomponents with or without the other. For example “A and/or B” is to betaken as specific disclosure of each of (i) A, (ii) B and (iii) A and B,just as if each is set out individually herein.

“Biophotonic” means the generation, manipulation, detection andapplication of photons in a biologically relevant context. In otherwords, biophotonic compositions exert their physiological effectsprimarily due to the generation and manipulation of photons.“Biophotonic composition” is a composition as described herein that maybe activated by light to produce photons for biologically relevantapplications.

“Topical” means as applied to body surfaces, such as the skin, mucousmembranes, vagina, oral cavity, internal surgical wound sites, and thelike.

Terms “chromophore,” “photoactivating agent,” and “photoactivator” areused herein interchangeably. A chromophore means a compound, whencontacted by light irradiation, is capable of absorbing the light. Thechromophore readily undergoes photoexcitation and can then transfer itsenergy to other molecules or emit it as light.

The term “oxidant” is intended to mean either a compound that readilytransfers oxygen atoms to and thus, oxidizes other compounds, or asubstance that gains electrons in a redox chemical reaction.

The term “chelating agent” is intended to mean a compound that bindsmetal ions, such as iron, cobalt, copper, manganese, and chromium ions,and facilitates their solvation in solution.

The term “healing factor” is intended to mean a compound that promotesor enhances the healing or regenerative process of a tissue.

The term “active oxygen species” is intended to mean chemically-reactivemolecules containing oxygen. Examples include, but are not limited to,oxygen ions and peroxides. They can be either inorganic or organic.Active oxygen species are highly reactive due to the presence ofunpaired valence shell electrons. They are also referred to as “reactiveoxygen,” “active oxygen,” or “reactive oxygen species.”

Features and advantages of the subject matter hereof will become moreapparent in light of the following detailed description of selectedembodiments, as illustrated in the accompanying figures. As will berealized, the subject matter disclosed herein is capable ofmodifications in various respects, all without departing from the scopeof the disclosed embodiments. Accordingly, the drawings and thedescription are to be regarded as illustrative in nature, and not asrestrictive and the full scope of the subject matter is set forth in theembodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the present disclosure will becomeapparent from the following detailed description, taken in combinationwith the appended drawings, in which:

FIG. 1 illustrates the Stokes' shift.

FIG. 2 illustrates the absorption and emission spectra of donor andacceptor chromophores. The spectral overlap between the absorptionspectrum of the acceptor chromophore and the emission spectrum of thedonor chromophore is also shown.

FIG. 3 is a schematic of a Jablonski diagram that illustrates thecoupled transitions involved between a donor emission and acceptorabsorbance.

FIG. 4 is a photograph showing an ear swab taken in a canine patient forbacteriology assessment before treatment.

FIG. 5 is a photograph showing an ear swab taken in a canine patient forcytological assessment.

FIG. 6 is a photograph showing measuring aural temperature bilaterallyin a canine patient before treatment.

FIG. 7 is a photograph of the external meatus in a canine patient beforetreatment.

FIGS. 8A-8B is a photograph showing preparation and application of thetherapeutic composition. FIG. 8A: preparation of the therapeuticcomposition by mixing chromophore and urea peroxide (UP); FIG. 8B:application of the therapeutic composition locally in a canine patient.

FIG. 9 is a photograph showing irradiation of the treatment area of acanine patient with a Bluephase® lamp, a source of actinic light.

FIG. 10 is a photograph showing an ear swab taken in a canine patientfor bacteriologic assessment after treatment.

FIG. 11 is a photograph showing measuring aural temperature bilaterallyin a canine patient after treatment.

FIG. 12 is a photograph of the external meatus in a canine patient aftertreatment.

FIGS. 13A-13D present graphs of various clinical score assessmentresults in the canine patients following biophotonic treatment, withFIG. 13A showing erythema results, FIG. 13B showing oedema/swellingresults, FIG. 13C showing erosion/ulceration results, and FIG. 13Dshowing exudate results.

FIG. 14 presents a graph of the total OTIS-3 score in canine patientsthat had received biophotonic treatment.

FIGS. 15A-15B present graphs of further clinical assessment criteria,with FIG. 15A showing pruritus results for canine patients, and FIG. 15Bshowing pain results for canine patients.

FIGS. 16A-16C present graphs of further clinical assessment criteria,with FIG. 16A showing aural temperature results in the canine patientpopulation receiving the biophotonic composition treatment, FIG. 16Bshowing the aural temperature results in the control canine patientpopulation, and FIG. 16C showing the treatment and control populations'aural temperature results in combination.

FIGS. 17A-17B present graphs of cytological assessment criteria, withFIG. 17A showing neutrophil results for canine patients, and FIG. 17Bshowing earwax/cerumen results for canine patients.

FIGS. 18A-18C presents graphs of further cytological assessmentcriteria, with FIG. 18A showing rod shaped bacteria results in thecanine patient population receiving the biophotonic compositiontreatment, FIG. 18B showing the coccoid bacterial population results inthe biophotonic composition treatment population, and FIG. 18C showingthe malassezia population results in the biophotonic compositiontreatment patient population.

FIGS. 19A-19B present graphs of bacteriology assessment criteria, withFIG. 19A showing a pie chart of the bacterial species and genera in theotitis-afflicted ear canals in canine patients pre-treatment, and FIG.19B showing a pie chart of the bacterial species and genera in theotitis-afflicted ear canals in canine patients post-treatment and duringfollow-up.

FIGS. 20A-20D present various clinical score assessment results of thecanine patients in the randomized controlled clinical trial described inthe Examples section of this disclosure. FIG. 20A shows the erythemaresults; FIG. 20B shows the oedema/swelling results; FIG. 20C shows theerosion/ulceration results; and FIG. 20D shows the exudate results.FIGS. 20A-20D were plotted to show the comparison of results betweenGroup I (Group QW), II (Group BW), and III (Group C) of the randomizedcontrolled clinical trial.

FIG. 21 presents the total OTIS-3 (otitis index scoring system) score incanine patients in Group I (Group QW), II (Group BW), and III (Group C)of the randomized controlled clinical trial described in the Examplessection of this disclosure, respectively.

FIGS. 22A-22B present graphs of clinical assessment criteria, with FIG.22A showing pruritus results for canine patients, and FIG. 22B showingpain results for canine patients. FIGS. 22A-22B were plotted to show thecomparison of results between Group I (Group QW), II (Group BW), and III(Group C) of the randomized controlled clinical trial.

FIGS. 23A-23B present graphs of cytological assessment criteria, withFIG. 23A showing neutrophil results for canine patients, and FIG. 23Bshowing earwax/cerumen results for canine patients. FIGS. 16A-16B wereplotted to show the comparison of results between Group I (Group QW), II(Group BW), and III (Group C) of the randomized controlled clinicaltrial.

FIGS. 24A-24C present graphs of cytological assessment criteria, withFIG. 24A showing rod shaped bacteria results in the canine patient, FIG.24B showing the coccoid bacterial population results in the caninepatient, and FIG. 24C showing the malassezia population results in thecanine patient. FIGS. 24A-24C were plotted to show the comparison ofresults between Group I (Group QW), II (Group BW), and III (Group C) ofthe randomized controlled clinical trial.

FIGS. 25A-25C present graphs of bacteriology assessment results. FIG.25A shows percentages of bacterial genus in Group I (Group QW)pre-treatment and after-treatment during follow-up; FIG. 25B showspercentages of bacterial genus in Group II (Group BW) pre-treatment andafter-treatment during follow-up; FIG. 25C shows percentages ofbacterial genus in Group III (Group C) pre-treatment and after-treatmentduring follow-up.

DETAILED DESCRIPTION

In some aspects, the disclosure provides a method of treating otitisexterna comprising: applying a biophotonic composition to a patient inneed thereof, wherein the biophotonic composition comprises at least oneoxidant and at least one chromophore capable of activating the oxidant;and exposing said biophotonic composition to actinic light for a timesufficient for said chromophore to cause activation of said oxidant. Incertain such aspects, the patient is a mammal, such as a human, afeline, or a canine. In certain such aspects, the otitis externa ischronic otitis externa. In some embodiments, the biophotoniccompositions of this disclosure are for topical use.

In some aspects, the disclosure provides for use of a biophotoniccomposition for the manufacture of a medicament for treating a patientafflicted with otitis externa, wherein said composition comprises: atleast one oxidant, and at least one chromophore capable of activatingthe oxidant; in association with a pharmacologically acceptable carrier.In certain such aspects, the patient is a mammal, such as a human, afeline or a canine. In certain such aspects, the otitis externa ischronic otitis externa.

In some aspects, the disclosure provides for use of a biophotoniccomposition for the treatment of a patient afflicted with otitisexterna, wherein said composition comprises: at least one oxidant; andat least one chromophore capable of activating the oxidant; inassociation with a pharmacologically acceptable carrier. In certain suchaspects, the patient is a mammal, such as a human, a feline or a canine.In certain such aspects, the otitis externa is chronic otitis externa.

Biophotonic Compositions

The present disclosure provides methods and uses comprising biophotoniccompositions for treating otitis externa. Biophotonic compositions arecompositions that are, in a broad sense, activated by light (e.g.,photons) of a specific wavelength. These compositions contain at leastone exogenous chromophore (e.g., a chromophore that is not naturallypresent in skin or tissue of the patient being treated), which isactivated by light and accelerates the dispersion of light energy, whichleads to light carrying on a therapeutic effect on its own, and/or tothe photochemical activation of other agents contained in thecomposition. The composition may comprise an agent which, when mixedwith a chromophore or combination of chromophores and subsequentlyactivated by light, can be photochemically activated which may lead tothe formation of oxygen radicals, such as singlet oxygen.

In some aspects, the disclosure provides a method of treating otitisexterna comprising: applying a biophotonic composition to a patient inneed thereof, wherein the biophotonic composition comprises at least oneoxidant and at least one chromophore capable of activating the oxidant;and exposing said biophotonic composition to actinic light for a timesufficient for said chromophore to cause activation of said oxidant.

When a chromophore absorbs a photon of a certain wavelength, it becomesexcited. This is an unstable condition and the molecule tries to returnto the ground state, giving away the excess energy. For somechromophores, it is favorable to emit the excess energy as light whentransforming back to the ground state. This process is calledfluorescence. The peak wavelength of the emitted fluorescence is shiftedtowards longer wavelengths compared to the absorption wavelengths(‘Stokes’ shift'). The emitted fluorescent energy can then betransferred to the other components of the composition or to a treatmentsite on to which the biophotonic composition is topically applied.Differing wavelengths of light may have different and complementarytherapeutic effects on tissue. Stokes' shift is illustrated in FIG. 1.

Without being bound to theory, it is thought that fluorescent lightemitted by photoactivated chromophores may have therapeutic propertiesdue to its femto-, pico- or nano-second emission properties which may berecognized by biological cells and tissues, leading to favorablebiomodulation. Furthermore, the emitted fluorescent light has a longerwavelength and hence a deeper penetration into the tissue than theactivating light. Irradiating tissue with such a broad range ofwavelengths, including in some embodiments the activating light whichpasses through the composition, may have different and complementaryeffects on the cells and tissues. Moreover, in some embodiments of thecomposition containing oxidants, micro-bubbling within the compositionhas been observed which may be associated with the generation of oxygenspecies by the photoactivated chromophores. This may have a physicalimpact on the tissue to which it is applied, for example by physicallydislodging biofilm and debridement of necrotic tissue or providing apressure stimulation. The biofilm can also be pre-treated with anoxygen-releasing agent to weaken the biofilm before treating with thecomposition of the present disclosure.

In certain embodiments, the biophotonic compositions of the presentdisclosure are substantially transparent/translucent and/or have highlight transmittance in order to permit light dissipation into andthrough the composition. In this way, the area of tissue under thecomposition can be treated both with the fluorescent light emitted bythe composition and the light irradiating the composition to activateit, which may benefit from the different therapeutic effects of lighthaving different wavelengths.

The % transmittance of the biophotonic composition can be measured inthe range of wavelengths from 250 nm to 800 nm using, for example, aPerkin-Elmer Lambda™ 9500 series UV-visible spectrophotometer.Alternatively, a Synergy™ HT spectrophotometer (BioTek Instrument, Inc.)can be used in the range of wavelengths from 380 nm to 900 nm.

Transmittance is calculated according to the following equation:

$A_{\lambda} = {{\log_{10}\frac{I_{0}}{I}} = {\log_{10}{\frac{1}{T}.}}}$where A is absorbance, T is transmittance, I₀ is intensity of radiationbefore passing through material, and I is intensity of light passingthrough material.

The values can be normalized for thickness. As stated herein, %transmittance (translucency) is as measured for a 2 mm thick sample at awavelength of 526 nm. It will be clear that other wavelengths can beused.

Embodiments of the biophotonic compositions of the present disclosureare for topical uses. The biophotonic composition can be in the form ofa semi-solid or viscous liquid, such as a gel, or are gel-like, andwhich have a spreadable consistency at room temperature (e.g., about20-25° C.), prior to illumination. By spreadable is meant that thecomposition can be topically applied to a treatment site at a thicknessof less than about 0.5 mm, from about 0.5 mm to about 3 mm, from about0.5 mm to about 2.5 mm, or from about 1 mm to about 2 mm. Spreadablecompositions can conform to a topography of a treatment site. This canhave advantages over a non-conforming material in that a better and/ormore complete illumination of the treatment site can be achieved and thecompositions are easy to apply and remove.

These compositions may be described based on the components making upthe composition. Additionally or alternatively, the compositions of thepresent disclosure have functional and structural properties and theseproperties may also be used to define and describe the compositions.Individual components of the composition of the present disclosure aredetailed below.

Oxidants

In some embodiments, the biophotonic compositions of the presentdisclosure comprise oxidants as a source of oxygen radicals. Forinstance, peroxide compounds are oxidants that contain the peroxy group(R—O—O—R), which is a chainlike structure containing two oxygen atoms,each of which is bonded to the other and a radical or some element.

In some embodiments, the biophotonic compositions of the presentdisclosure comprise an oxidant selected from, but not limited to,hydrogen peroxide, urea hydrogen peroxide, benzoyl peroxide, peroxyacids, or alkali metal percarbonates.

Suitable oxidants for the biophotonic compositions of the presentdisclosure include, but are not limited to:

Hydrogen peroxide (H₂O₂) is the starting material to prepare organicperoxides. H₂O₂ is a powerful oxidizing agent, and the unique propertyof hydrogen peroxide is that it breaks down into water and oxygen anddoes not form any persistent, toxic residual compound. Hydrogen peroxidefor use in this composition can be used in a gel, for example with 6%hydrogen peroxide by weight of the total composition. A suitable rangeof concentration over which hydrogen peroxide can be used in acomposition of the present disclosure is less than about 12% by weightof the total compositions. In some embodiments, hydrogen peroxide ispresent in an amount from about 0.1% to about 12%, from about 1% toabout 12%, from about 3.5% to about 12%, from about 3.5% to about 6%, orfrom about 0.1% to about 6% by weight of the total composition.

Urea hydrogen peroxide (also known as urea peroxide, carbamide peroxideor percarbamide) is soluble in water and contains about 36% hydrogenperoxide. Carbamide peroxide for use in this composition can be used asa gel, for example with about 16% carbamide peroxide that representsabout 5.6% hydrogen peroxide. A suitable range of concentration overwhich urea peroxide can be used in a composition of the presentdisclosure is less than about 36% by weight of the total composition. Insome embodiments, urea peroxide is present in an amount from about 0.3%to about 36%, such as from about 3% to about 36%, from about 10% toabout 36%, from about 3% to about 16%, from about 3% to about 9%, orfrom about 0.3% to about 16% by weight of the total composition. In someembodiments, urea peroxide is present in an amount of about 2% by weightof the total composition. In some embodiments, urea peroxide is presentin an amount of about 3% by weight of the total composition. In someembodiments, urea peroxide is present in an amount of about 6% by weightof the total composition. In some embodiments, urea peroxide is presentin an amount of about 8% by weight of the total composition. In someembodiments, urea peroxide is present in an amount of about 9% of thetotal composition. In some embodiments, urea peroxide is present in anamount of about 12% by weight of the total composition. Urea peroxidebreaks down to urea and hydrogen peroxide in a slow-release fashion thatcan be accelerated with heat or photochemical reactions. The releasedurea (i.e., carbamide, (NH₂)₂CO) is highly soluble in water and is apowerful protein denaturant. It increases solubility of some proteinsand enhances rehydration of the skin and/or mucosa.

Benzoyl peroxide consists of two benzoyl groups (benzoic acid with the Hof the carboxylic acid removed) joined by a peroxide group. The releasedperoxide groups are effective at killing bacteria. Benzoyl peroxide alsopromotes skin turnover and clearing of pores. Benzoyl peroxide breaksdown to benzoic acid and oxygen upon contact with skin, neither of whichis toxic. A suitable range of concentration over which benzoyl peroxidecan be used in the present composition is less than about 10% by weightof the total composition, such as from about 1% to 10%, from about 1% to8%, from about 2.5% to about 5%. In some embodiments, benzoyl peroxideis present in an amount from about 1% to about 10%, or from about 1% toabout 8%, or from about 2.5% to about 5% by weight of the totalcomposition.

Suitable oxidants may also include peroxy acids and alkali metalpercarbonates, but the inclusion of any other forms of peroxides (e.g.,organic or inorganic peroxides) should be avoided due to their increasedtoxicity and their unpredictable reaction with the photodynamic energytransfer.

Chromophores/Photoactivators

In some embodiments, the biophotonic topical compositions of the presentdisclosure comprise one or more chromophores, which can be consideredexogenous, e.g., are not naturally present in skin or tissue. When abiophotonic composition of the present disclosure is illuminated withlight, the chromophore(s) are excited to a higher energy state. When thechromophore(s)' electrons return to a lower energy state, they emitphotons with a lower energy level, thus causing the emission of light ofa longer wavelength (Stokes' shift). In the proper environment, some ofthis energy release is transferred to oxygen and causes the formation ofoxygen radicals, such as singlet oxygen.

Suitable chromophores for the biophotonic compositions of the disclosurecan be fluorescent dyes (or stains), although other dye groups or dyes(biological and histological dyes, food colorings, carotenoids,naturally occurring fluorescent and other dyes) can also be used.

In some embodiments, the biophotonic topical composition of the presentdisclosure comprises a chromophore which undergoes partial or completephotobleaching upon application of light. By photobleaching is meant aphotochemical destruction of the chromophore which can generally becharacterized as a visual loss of color or loss of fluorescence.

In some embodiments, the chromophore absorbs at a wavelength in therange of the visible spectrum, such as at a wavelength of from about 380to about 800 nm, such as from about 380 to about 700 nm, or from about380 to about 600 nm. In some embodiments, the chromophore absorbs at awavelength of from about 200 to about 800 nm, such as from about 200 toabout 700 nm, from about 200 to about 600 nm, or from about 200 to about500 nm. In some embodiments, the chromophore absorbs at a wavelength offrom about 200 to about 600 nm. In some embodiments, the chromophoreabsorbs light at a wavelength of from about 200 to about 300 nm, fromabout 250 to about 350 nm, from about 300 to about 400 nm, from about350 to about 450 nm, from about 400 to about 500 nm, from about 400 toabout 600 nm, from about 450 to about 650 nm, from about 600 to about700 nm, from about 650 to about 750 nm, or from about 700 to about 800nm.

In some embodiments, the chromophore or combination of chromophores ispresent in an amount of from about 0.001 to about 40% by weight of thetotal composition. In some embodiments, the chromophore or combinationof chromophores is present in an amount of from about 0.005 to about 2%,from about 0.01 to about 1%, from about 0.01 to about 2%, from about0.05 to about 1%, from about 0.05 to about 2%, from about 0.1 to about1%, from about 0.1 to about 2%, from about 1-5%, from about 2.5 to about7.5%, from about 5 to about 10%, from about 7.5 to about 12.5%, fromabout 10 to about 15%, from about 12.5 to about 17.5%, from about 15 to20%, from about 17.5 to about 22.5%, from about 20 to about 25%, fromabout 22.5 to about 27.5%, from about 25 to about 30%, from about 27.5to about 32.5%, from about 30 to about 35%, from about 32.5 to about37.5%, or from about 35 to about 40% by weight of the total composition.In some embodiments, the chromophore or combination of chromophores ispresent in an amount of at least about 0.2% by weight of the totalcomposition.

In some embodiments, the chromophore or combination of chromophores ispresent in an amount of 0.001% to 40% by weight of the totalcomposition. In some embodiments, the chromophore or combination ofchromophores is present in an amount of from 0.005 to 2%, from 0.01 to1%, from 0.01% to 2%, from 0.05% to 1%, from 0.05-2%, from 0.1% to 1%,from 0.1% to 2%, from 1 to 5%, from 2.5 to 7.5%, from 5 to 10%, from7.5% to 12.5%, from 10% to 15%, from 12.5% to 17.5%, from 15% to 20%,from 17.5% to 22.5%, from 20% to 25%, from 22.5% to 27.5%, from 25% to30%, from 27.5% to 32.5%, from 30% to 35%, from 32.5% to 37.5%, or from35% to 40% by weight of the total composition. In some embodiments, thechromophore or combination of chromophores is present in an amount of atleast 0.2% by weight of the total composition.

It will be appreciated to those skilled in the art that opticalproperties of a particular chromophore may vary depending on thechromophore's surrounding medium. Therefore, as used herein, aparticular chromophore's absorption and/or emission wavelength (orspectrum) corresponds to the wavelengths (or spectra) measured in abiophotonic composition of the present disclosure.

In some embodiments, the biophotonic compositions disclosed herein mayinclude at least one additional chromophore. Combining chromophores mayincrease photo-absorption by the combined dye molecules and enhanceabsorption and photo-biomodulation selectivity. This creates multiplepossibilities of generating new photosensitive, and/or selectivechromophores mixtures.

When such multi-chromophore compositions are illuminated with light,energy transfer can occur between the chromophores. This process, knownas resonance energy transfer, is a photophysical process through whichan excited ‘donor’ chromophore (also referred to herein as firstchromophore) transfers its excitation energy to an ‘acceptor’chromophore (also referred to herein as second chromophore). Theefficiency and directedness of resonance energy transfer depends on thespectral features of donor and acceptor chromophores. In particular, theflow of energy between chromophores is dependent on a spectral overlapreflecting the relative positioning and shapes of the absorption andemission spectra. For energy transfer to occur the emission spectrum ofthe donor chromophore overlap with the absorption spectrum of theacceptor chromophore (FIG. 2).

Energy transfer manifests itself through decrease or quenching of thedonor emission and a reduction of excited state lifetime accompaniedalso by an increase in acceptor emission intensity. FIG. 3 is aJablonski diagram that illustrates the coupled transitions involvedbetween a donor emission and acceptor absorbance.

To enhance the energy transfer efficiency, the donor chromophore shouldhave good abilities to absorb photons and emit photons. Furthermore, itis thought that the more overlap there is between the donorchromophore's emission spectra and the acceptor chromophore's absorptionspectra, the better a donor chromophore can transfer energy to theacceptor chromophore.

In some embodiments, the biophotonic topical composition of the presentdisclosure further comprises an acceptor, or a second, chromophore. Insome embodiments, the donor, or first, chromophore has an emissionspectrum that overlaps at least about 80%, about 70%, about 60%, about50%, about 40%, about 30%, about 20%, or about 10% with an absorptionspectrum of the second chromophore. In some embodiments, the firstchromophore has an emission spectrum that overlaps at least about 20%with an absorption spectrum of the second chromophore. In someembodiments, the first chromophore has an emission spectrum thatoverlaps at least 1-10%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%,35-45%, 50-60%, 55-65% or 60-70% with an absorption spectrum of thesecond chromophore.

Percentage (%) spectral overlap, as used herein, means the % overlap ofa donor chromophore's emission wavelength range with an acceptorchromophore's absorption wavelength rage, measured at spectral fullwidth quarter maximum (FWQM). For example, FIG. 2 shows the normalizedabsorption and emission spectra of donor and acceptor chromophores. Thespectral FWQM of the acceptor chromophore's absorption spectrum is fromabout 60 nm (about 515 nm to about 575 nm). The overlap of the donorchromophore's spectrum with the absorption spectrum of the acceptorchromophore is about 40 nm (from 515 nm to about 555 nm). Thus, the %overlap can be calculated as 40 nm/60 nm×100=66.6%.

In some embodiments, the second chromophore absorbs at a wavelength inthe range of the visible spectrum. In some embodiments, the secondchromophore has an absorption wavelength that is relatively longer thanthat of the first chromophore within the range of about 50-250 nm, about25-150 nm or about 10-100 nm.

As discussed above, the application of light to the compositions of thepresent disclosure can result in a cascade of energy transfer betweenthe chromophores. In some embodiments, such a cascade of energy transferprovides photons that penetrate the epidermis, dermis and/or mucosa ofthe target tissue.

In some embodiments, the chromophore or chromophores are selected suchthat their emitted fluorescent light, on photoactivation, is within oneor more of the green, yellow, orange, red and infrared portions of theelectromagnetic spectrum, for example having a peak wavelength withinthe range of about 490 nm to about 800 nm. In some embodiments, theemitted fluorescent light has a power density of between 0.005 to about10 mW/cm² or about 0.5 to about 5 mW/cm².

Suitable chromophores useful in the biophotonic topical compositions,methods, and uses of the present disclosure include, but are not limitedto the following:

Xanthene Derivatives

The xanthene derivative dyes have been used and tested for a long timeworldwide. They display low toxicity and increased fluorescence. Thexanthene group consists of three sub-groups: a) the fluorenes; b)fluorones; and c) the rhodoles, any of which may be suitable for thebiophotonic compositions, methods, and uses of the present disclosure.

The fluorenes group comprises the pyronines (e.g., pyronine Y and B) andthe rhodamines (e.g., rhodamine B, G and WT). Depending on theconcentration used, both pyronines and rhodamines may be toxic and theirinteraction with light may lead to increased toxicity. Similar effectsare known to occur for the rhodole dye group.

The fluorone group comprises the fluorescein dye and the fluoresceinderivatives.

Fluorescein is a fluorophore commonly used in microscopy with anabsorption maximum of about 494 nm and an emission maximum of about 521nm. The disodium salt of fluorescein is known as D&C Yellow 8. It hasvery high fluorescence but photodegrades quickly. In the presentcomposition, mixtures of fluorescein with other photoactivators such asindocyanin green and/or saffron red powder will confer increasedphotoabsorption to these other compounds.

The eosins group comprises Eosin Y (tetrabromofluorescein, acid red 87,D&C Red 22), a chromophore with an absorption maximum of from about 514to about 518 nm that stains the cytoplasm of cells, collagen, musclefibers and red blood cells intensely red; and Eosin B (acid red 91,eosin scarlet, dibromo-dinitrofluorescein), with the same stainingcharacteristics as Eosin Y. Eosin Y and Eosin B are collectivelyreferred to as “Eosin,” and use of the term “Eosin” refers to eitherEosin Y, Eosin B or a mixture of both. Eosin Y, Eosin B, or a mixture ofboth can be used because of their sensitivity to the light spectra used:broad spectrum blue light, blue to green light and green light.

In some embodiments, the composition includes in the range of less thanabout 12% by weight of the total composition of at least one of Eosin Bor Eosin Y or a combination thereof. In some embodiments, at least oneof Eosin B or Eosin Y or a combination thereof is present from about0.001% to about 12%, or between about 0.01% and about 1.2%, or fromabout 0.01% to about 0.5%, or from about 0.01% to about 0.05%, or fromabout 0.1% to about 0.5%, or from about 0.5% to about 0.8% by weight ofthe total composition. In some embodiments, at least one of Eosin B orEosin Y or a combination thereof is present is an amount of about 0.005%by weight of the total composition. In some embodiments, at least one ofEosin B or Eosin Y or a combination thereof is present is an amount ofabout 0.01% by weight of the total composition. In some embodiments, atleast one of Eosin B or Eosin Y or a combination thereof is present isan amount of about 0.02% by weight of the total composition. In someembodiments, at least one of Eosin B or Eosin Y or a combination thereofis present is an amount of about 0.05% by weight of the totalcomposition. In some embodiments, at least one of Eosin B or Eosin Y ora combination thereof is present is an amount of about 0.1% by weight ofthe total composition. In some embodiments, at least one of Eosin B orEosin Y or a combination thereof is present is an amount of about 0.2%by weight of the total composition. In some embodiments, at least one ofEosin B or Eosin Y or a combination thereof is present is an amount ofat least about 0.2% by weight of the total composition but less thanabout 1.2% by weight of the total composition. In some embodiments, atleast one of Eosin B or Eosin Y or a combination thereof is present isan amount of at least about 0.01% by weight of the total composition butless than about 12% by weight of the total composition.

In some embodiments, the composition includes in the range of less than12% by weight of the total composition of at least one of Eosin B orEosin Y or a combination thereof. In some embodiments, at least one ofEosin B or Eosin Y or a combination thereof is present from 0.001% to12%, or from 0.01% to 1.2%, or from 0.01% to 0.5%, or from 0.1% to 0.5%,or from 0.5% to 0.8%, or from 0.01% to 0.05%, by weight of the totalcomposition. In some embodiments, at least one of Eosin B or Eosin Y ora combination thereof is present is an amount of 0.005% by weight of thetotal composition. In some embodiments, at least one of Eosin B or EosinY or a combination thereof is present is an amount of 0.01% by weight ofthe total composition. In some embodiments, at least one of Eosin B orEosin Y or a combination thereof is present is an amount of 0.02% byweight of the total composition. In some embodiments, at least one ofEosin B or Eosin Y or a combination thereof is present is an amount of0.05% by weight of the total composition. In some embodiments, at leastone of Eosin B or Eosin Y or a combination thereof is present is anamount of 0.1% by weight of the total composition. In some embodiments,at least one of Eosin B or Eosin Y or a combination thereof is presentis an amount of 0.2% by weight of the total composition. In someembodiments, at least one of Eosin B or Eosin Y or a combination thereofis present is an amount of at least 0.2% by weight of the totalcomposition but less than 1.2% by weight of the total composition. Insome embodiments, at least one of Eosin B or Eosin Y or a combinationthereof is present is an amount of at least 0.01% by weight of the totalcomposition but less than 12% by weight of the total composition.

Phloxine B (2,4,5,7 tetrabromo 4,5,6,7,tetrachlorofluorescein, D&C Red28, acid red 92) is a red dye derivative of fluorescein which is usedfor disinfection and detoxification of waste water throughphotooxidation. It has an absorption maximum of 535-548 nm. It is alsoused as an intermediate for making photosensitive dyes and drugs.

Erythrosine B, or simply Erythrosine or Erythrosin (acid red 51,tetraiodofluorescein) is a cherry-pink, coal-based fluorine food dyeused as a biological stain, and a biofilm and dental plaque disclosingagent, with a maximum absorbance of 524-530 nm in aqueous solution. Itis subject to photodegradation. Erythrosine is also used in someembodiments due to its photosensitivity to the light spectra used andits ability to stain biofilms. In embodiments, the composition includesin the range of less than about 2% by weight Erythrosine B. In someembodiments, Erythrosine B is present in an amount from about 0.005 toabout 2%, or from about 0.005% to about 1%, or about 0.01% to about 1%by weight of the total composition. In some embodiments, Erythrosine Bis present in an amount of about 0.005% and about 0.15% by weight of thetotal composition.

Rose Bengal (4,5,6,7 tetrachloro 2,4,5,7 tetraiodofluorescein, acid red94) is a bright bluish-pink fluorescein derivative with an absorptionmaximum of 544-549 nm, that has been used as a dye, biological stain anddiagnostic aid. Rose Bengal is also used in synthetic chemistry togenerate singlet oxygen from triplet oxygen.

Merbromine (mercurochrome) is an organo-mercuric disodium salt offluorescein with an absorption maximum of 508 nm. It is used as anantiseptic.

Azo Dyes

The azo (or diazo-) dyes share the N—N group, called azo the group. Theyare used mainly in analytical chemistry or as food colorings and are notfluorescent. Suitable azo dyes for the compositions, methods, and usesof the disclosure include: Methyl violet, neutral red, para red (pigmentred 1), amaranth (Azorubine S), Carmoisine (azorubine, food red 3, acidred 14), allura red AC (FD&C 40), tartrazine (FD&C Yellow 5), orange G(acid orange 10), Ponceau 4R (food red 7), methyl red (acid red 2), andmurexide-ammonium purpurate.

Biological Stains

Dye molecules commonly used in staining protocols for biologicalmaterials can also be used as photoactivators for the compositions,methods, and uses of the disclosure. Suitable biological stains include,but not limited to:

Saffranin (Saffranin 0, basic red 2) is an azo-dye and is used inhistology and cytology. It is a classic counter stain in a Gram stainprotocol.

Fuchsin (basic or acid) (rosaniline hydrochloride) is a magentabiological dye that can stain bacteria and has been used as anantiseptic. It has an absorption maximum of 540-555 nm.

3,3′-dihexylocarbocyanine iodide (DiOC6) is a fluorescent dye used forstaining the endoplasmic reticulum, vesicle membranes and mitochondriaof cells. It shows photodynamic toxicity; when exposed to blue light,has a green fluorescence.

Carminic acid (acid red 4, natural red 4) is a red glucosidalhydroxyanthrapurin naturally obtained from cochineal insects.

Indocyanin green (ICG) is used as a diagnostic aid for blood volumedetermination, cardiac output, or hepatic function. ICG binds stronglyto red blood cells and when used in mixture with fluorescein, itincreases the absorption of blue to green light.

Carotenoids

Carotenoid dyes are also photoactivators that are useful in thecompositions, methods, and uses of the disclosure.

Saffron red powder is a natural carotenoid-containing compound. Saffronis a spice derived from crocus sativus. It is characterized by a bittertaste and iodoform or hay-like fragrance; these are caused by thecompounds picrocrocin and saffranal. It also contains the carotenoid dyecrocin that gives its characteristic yellow-red color.

Saffron contains more than 150 different compounds, many of which arecarotenoids: mangicrocin, reaxanthine, lycopene, and various α andβ-carotenes, which show good absorption of light and beneficialbiological activity. Also saffron can act as both a photon-transferagent and a healing factor. Saffron color is primarily the result ofa-crocin (8,8 diapo-8,8-carotenoid acid). Dry saffron red powder ishighly sensitive to fluctuating pH levels and rapidly breaks downchemically in the presence of light and oxidizing agents. It is moreresistant to heat. Data show that saffron has anticarcinogenic,immunomodulating and antioxidant properties. For absorbance, the crocinspecific photon wavelength is 440 nm (blue light). It has a deep redcolour and forms crystals with a melting point of 186° C. When dissolvedin water, it forms an orange solution.

Crocetin, another compound of saffron, was found to express anantilipidemic action and promote oxygen penetration in differenttissues. More specifically, an increased oxygenation of the endothelialcells of the capillaries was observed. Additionally, an increase of theoxygenation of muscles and cerebral cortex was observed and led to animproved survival rate in laboratory animals with induced hemorrhagicshock or emphysema.

Anatto, a spice, contains as main constituent (70-80%) the carotenoidbixin which displays relevant antioxidative properties. β-carotene, alsodisplays suitable characteristics.

Fucoxanthine is a constituent of brown algae with a pronounced abilityfor photosensitization of redox reactions.

Chlorophyll Dyes

Exemplary chlorophyll dyes that are useful in the compositions, methods,and uses of the disclosure, include but are not limited to chlorophylla, chlorophyll b, oil soluble chlorophyll, bacteriochlorophyll a,bacteriochlorophyll b, bacteriochlorophyll c, bacteriochlorophyll d,protochlorophyll, protochlorophyll a, amphiphilic chlorophyll derivative1, and amphiphilic chlorophyll derivative 2.

In some aspects of the disclosure, the one or more chromophores of thebiophotonic composition disclosed herein can be independently selectedfrom any of Acid black 1, Acid blue 22, Acid blue 93, Acid fuchsin, Acidgreen, Acid green 1, Acid green 5, Acid magenta, Acid orange 10, Acidred 26. Acid red 29, Acid red 44, Acid red 51, Acid red 66, Acid red 87,Acid red 91, Acid red 92, Acid red 94, Acid red 101, Acid red 103, Acidroseine, Acid rubin, Acid violet 19, Acid yellow 1, Acid yellow 9, Acidyellow 23, Acid yellow 24, Acid yellow 36, Acid yellow 73, Acid yellowS, Acridine orange, Acriflavine, Alcian blue, Alcian yellow, Alcoholsoluble eosin, Alizarin, Alizarin blue 2RC, Alizarin carmine, Alizarincyanin BBS, Alizarol cyanin R, Alizarin red S, Alizarin purpurin,Aluminon, Amido black 10B, Amidoschwarz, Aniline blue WS, Anthraceneblue SWR, Auramine O, Azocannine B, Azocarmine G, Azoic diazo 5, Azoicdiazo 48, Azure A, Azure B, Azure C, Basic blue 8, Basic blue 9, Basicblue 12, Basic blue 15, Basic blue 17, Basic blue 20, Basic blue 26,Basic brown 1, Basic fuchsin, Basic green 4, Basic orange 14, Basic red2 (Saffranin O), Basic red 5, Basic red 9, Basic violet 2, Basic violet3, Basic violet 4, Basic violet 10, Basic violet 14, Basic yellow 1,Basic yellow 2, Biebrich scarlet, Bismarck brown Y, Brilliant crystalscarlet 6R, Calcium red, Carmine, Carminic acid (acid red 4), Celestineblue B, China blue, Cochineal, Celestine blue, Chrome violet CG,Chromotrope 2R, Chromoxane cyanin R, Congo corinth, Congo red, Cottonblue, Cotton red, Croceine scarlet, Crocin, Crystal ponceau 6R, Crystalviolet, Dahlia, Diamond green B, DiOC6, Direct blue 14, Direct blue 58,Direct red, Direct red 10, Direct red 28, Direct red 80, Direct yellow7, Eosin B, Eosin Bluish, Eosin, Eosin Y, Eosin yellowish, Eosinol, Eriegarnet B, Eriochrome cyanin R, Erythrosin B, Ethyl eosin, Ethyl green,Ethyl violet, Evans blue, Fast blue B, Fast green FCF, Fast red B, Fastyellow, Fluorescein, Food green 3, Gallein, Gallamine blue, Gallocyanin,Gentian violet, Haematein, Haematine, Haematoxylin, Helio fast rubinBBL, Helvetia blue, Hematein, Hematine, Hematoxylin, Hoffman's violet,Imperial red, Indocyanin green, Ingrain blue, Ingrain blue 1, Ingrainyellow 1, INT, Kermes, Kermesic acid, Kernechtrot, Lac, Laccaic acid,Lauth's violet, Light green, Lissamine green SF, Luxol fast blue,Magenta 0, Magenta I, Magenta II, Magenta III, Malachite green,Manchester brown, Martius yellow, Merbromin, Mercurochrome, Metanilyellow, Methylene azure A, Methylene azure B, Methylene azure C,Methylene blue, Methyl blue, Methyl green, Methyl violet, Methyl violet2B, Methyl violet 10B, Mordant blue 3, Mordant blue 10, Mordant blue 14,Mordant blue 23, Mordant blue 32, Mordant blue 45, Mordant red 3,Mordant red 11, Mordant violet 25, Mordant violet 39 Naphthol blueblack, Naphthol green B, Naphthol yellow S, Natural black 1, Naturalred, Natural red 3, Natural red 4, Natural red 8, Natural red 16,Natural red 25, Natural red 28, Natural yellow 6, NBT, Neutral red, Newfuchsin, Niagara blue 3B, Night blue, Nile blue, Nile blue A, Nile blueoxazone, Nile blue sulphate, Nile red, Nitro BT, Nitro blue tetrazolium,Nuclear fast red, Oil red O, Orange G, Orcein, Pararosanilin, PhloxineB, phycobilins, Phycocyanins, Phycoerythrins. Phycoerythrincyanin (PEC),Phthalocyanines, Picric acid, Ponceau 2R, Ponceau 6R, Ponceau B, Ponceaude Xylidine, Ponceau S, Primula, Purpurin, Pyronin B, Pyronin G, PyroninY, Rhodamine B, Rosanilin, Rose bengal, Saffron, Safranin O, Scarlet R,Scarlet red, Scharlach R, Shellac, Sirius red F3B, Solochrome cyanin R,Soluble blue, Solvent black 3, Solvent blue 38, Solvent red 23, Solventred 24, Solvent red 27, Solvent red 45, Solvent yellow 94, Spiritsoluble eosin, Sudan III, Sudan IV, Sudan black B, Sulfur yellow S,Swiss blue, Tartrazine, Thioflavine S, Thioflavine T, Thionin, Toluidineblue, Toluyline red, Tropaeolin G, Trypaflavine, Trypan blue, Uranin,Victoria blue 4R, Victoria blue B, Victoria green B, Water blue I, Watersoluble eosin, Xylidine ponceau, or Yellowish eosin.

Chromophores can be selected, for example, on their emission wavelengthproperties in the case of fluorophores, on the basis of their energytransfer potential, their ability to generate reactive oxygen species,or their antimicrobial effect.

In some embodiments, the biophotonic compositions of this disclosurecomprises Eosin Y as a first chromophore. In some embodiments, thecomposition comprises Eosin Y as a first chromophore and any one or moreof Rose Bengal, Erythrosin, Phloxine B as a second chromophore. It isbelieved that these combinations have a synergistic effect as Eosin Ycan transfer energy to Rose Bengal, Erythrosin or Phloxine B whenactivated. This transferred energy is then emitted as fluorescence or byproduction of reactive oxygen species. This absorbed and re-emittedlight is thought to be transmitted throughout the composition, and alsoto be transmitted into the site of treatment.

In some embodiments, the biophotonic compositions of this disclosurecomprise the following synergistic combinations: Eosin Y andFluorescein; Fluorescein and Rose Bengal; Erythrosine in combinationwith one or more of Eosin Y, Rose Bengal or Fluorescein; or Phloxine Bin combination with one or more of Eosin Y, Rose Bengal, Fluorescein andErythrosine. Other synergistic chromophore combinations may also besuitable for the biophotonic compositions of this disclosure.

By means of synergistic effects of the chromophore combinations in thecomposition, chromophores which cannot normally be activated by anactivating light (such as a blue light from an LED) can be activatedthrough energy transfer from chromophores which are activated by theactivating light. In this way, the different properties ofphotoactivated chromophores can be harnessed and tailored according tothe cosmetic or the medical therapy required.

For example, Rose Bengal can generate a high yield of singlet oxygenwhen photoactivated in the presence of molecular oxygen, however, it hasa low quantum yield in terms of emitted fluorescent light. Rose Bengalhas a peak absorption at approximately 540 nm; so it is normallyactivated by green light. Eosin Y has a high quantum yield and can beactivated by blue light. By combining Rose Bengal with Eosin Y, oneobtains a composition which can emit therapeutic fluorescent light andgenerate singlet oxygen when activated by blue light. In this case, theblue light photoactivates Eosin Y, which transfers some of its energy toRose Bengal and emits some energy as fluorescence.

Chromophore combinations can also have a synergistic effect in terms oftheir photoactivated state. In some embodiments, two chromophores may beused, one of which emits fluorescent light when activated in the blueand green range, and the other which emits fluorescent light in the red,orange and yellow range, thereby complementing each other andirradiating the target tissue with a broad wavelength of light havingdifferent depths of penetration into target tissue and differenttherapeutic effects.

Healing Factors

According to some embodiments, the biophotonic compositions of thepresent disclosure may further comprise one or more healing factors.Healing factors include compounds that promote or enhance the healing orregenerative process of the tissues on the application site of thecomposition. During the photoactivation of the composition, there is anincrease of the absorption of molecules at the treatment site. Anaugmentation in the blood flow at the site of treatment is observed foran extended period of time. An increase in the lymphatic drainage and apossible change in the osmotic equilibrium due to the dynamicinteraction of the free radical cascades can be enhanced or evenfortified with the inclusion of healing factors. In some embodiments,the biophotonic compositions of the present disclosure comprises one ormore healing factors selected from, but not limited to, hyaluronic acid,glucosamine, allantoin, or saffron.

Suitable healing factors for the biophotonic compositions, methods anduses of the present disclosure include, but are not limited to:

Hyaluronic acid (hyaluronan or hyaluronate) is a non-sulfatedglycosaminoglycan, distributed widely throughout connective, epithelialand neural tissues. It is one of the primary components of theextracellular matrix, and contributes significantly to cellproliferation and migration. Hyaluronan is a major component of theskin, where it is involved in tissue repair. While it is abundant inextracellular matrices, it contributes to tissue hydrodynamics, movementand proliferation of cells and participates in a wide number of cellsurface receptor interactions, notably those including primary receptorCD44. The hyaluronidase enzymes degrade hyaluronan and there are atleast seven types of hyaluronidase-like enzymes in humans, several ofwhich are tumor suppressors. The degradation products of hyaluronicacid, the oligosaccharides and the very-low molecular weight hyaluronicacid, exhibit pro-angiogenic properties. In addition, recent studiesshow that hyaluronan fragments, but not the native high molecular massof hyaluronan, can induce inflammatory responses in macrophages anddendritic cells in tissue injury. Hyaluronic acid is well suited tobiological applications targeting the skin. Due to its highbiocompatibility, it is used to stimulate tissue regeneration. Currentstudies evidenced hyaluronic acid appearing in the early stages ofhealing to physically create room for white blood cells that mediate theimmune response. It is used in the synthesis of biological scaffolds forwound healing applications and in wrinkle treatment. In certainembodiments, the composition includes hyaluronic acid in the range ofless than about 2% by weight of the total composition hyaluronic acid.In some embodiments, hyaluronic acid is present in an amount from about0.001% to about 2%, or from about 0.002% to about 2%, or from about0.002% to about 1% by weight of the total composition.

Glucosamine is one of the most abundant monosaccharides in human tissuesand a precursor in the biological synthesis of glycosylated proteins andlipids. It is commonly used in the treatment of osteoarthritis. Thecommon form of glucosamine used is its sulfate salt. Glucosamine shows anumber of effects, including anti-inflammatory activity, stimulation ofthe synthesis of proteoglycans and the synthesis of proteolytic enzymes.A suitable range of concentration over which glucosamine can be used inthe present composition is from less than about 5% by weight of thetotal composition. In some embodiments, glucosamine is present in anamount from about 0.0001% to about 5%, or from about 0.0001% to about3%, or from about 0.001% to about 3%, or from about 0.001% to about 1%,or from about 0.01% to about 1%, or from about 1% to about 3% by weightof the total composition.

Allantoin is a diureide of glyosilic acid. It has keratolytic effect,increases the water content of the extracellular matrix, enhances thedesquamation of the upper layers of dead (apoptotic) skin cells, andpromotes skin proliferation and wound healing. In certain embodiments,the composition includes in the range of less than about 1% by weight ofthe total composition allantoin. In some embodiments, allantoin ispresent in an amount of from about 0.001% to about 1%, or from about0.002% to about 1%, or from about 0.02% to about 1%, or from about 0.02%to about 0.5% by weight of the total composition.

Saffron can act as both a photon-transfer agent and a healing factor.

Chelating Agents

In some embodiments, the biophotonic compositions of the presentdisclosure may further comprise one or more chelating factors. Chelatingagents can be included to promote smear layer removal in closed pocketsand difficult to reach lesions. Chelating agents act as a metal ionquencher and as a buffer. In some embodiments, the biophotoniccompositions of the present disclosure comprise a chelating factorselected from, but not limited to, ethylenediaminotetraacetic acid(EDTA) or ethylene glycol tetraacetic acid (EGTA.

Suitable chelating agents for the biophotonic compositions, methods anduses of this disclosure include, but are not limited to:

Ethylenediaminotetraacetic Acid (EDTA)

Ethylenediaminotetraacetic acid (EDTA) is an amino acid and is used tosequester di- and trivalent metal ions. EDTA binds to metals via fourcarboxylate and two amine groups. EDTA forms especially strong complexeswith Mn(III), Fe(III), Cu(III), Co(III). It is used to buffer solutions.

Ethylene Glycol Tetraacetic Acid (EGTA)

Ethylene glycol tetraacetic acid (EGTA) is related to EDTA, but with amuch higher affinity for calcium than magnesium ions. It is useful formaking buffer solutions that resemble the environment inside livingcells.

Gelling Agents

In some embodiments, the biophotonic compositions of the presentdisclosure may further comprise one or more gelling agents. The gellingagent may be an agent capable of forming a cross-linked matrix,including physical and/or chemical cross-links. The gelling agent can bebiocompatible, and may be biodegradable. In some embodiments, thegelling agent is able to form a hydrogel or a hydrocolloid. Anappropriate gelling agent is one that can form a viscous liquid or asemisolid. In some embodiments, the gelling agent and/or the compositionhas appropriate light transmission properties. It is also important toselect a gelling agent which will allow biophotonic activity of thechromophore(s). For example, some chromophores require a hydratedenvironment in order to fluoresce. The gelling agent may be able to forma gel by itself or in combination with other ingredients such as wateror another gelling agent, or when applied to a treatment site, or whenilluminated with light.

The gelling agent according to various embodiments of the presentdisclosure may include, but not be limited to, polyalkylene oxides,particularly polyethylene glycol and poly(ethylene oxide)-poly(propyleneoxide) copolymers, including block and random copolymers; polyols suchas glycerol, polyglycerol (particularly highly branched polyglycerol),propylene glycol and trimethylene glycol substituted with one or morepolyalkylene oxides, e.g., mono-, di- and tri-polyoxyethylated glycerol,mono- and di-polyoxy-ethylated propylene glycol, and mono- anddi-polyoxyethylated trimethylene glycol; polyoxyethylated sorbitol,polyoxyethylated glucose; acrylic acid polymers and analogs andcopolymers thereof, such as polyacrylic acid per se, polymethacrylicacid, poly(hydroxyethylmethacrylate), poly(hydroxyethylacrylate),poly(methylalkylsulfoxide methacrylate), poly(methylalkylsulfoxideacrylate) and copolymers of any of the foregoing, and/or with additionalacrylate species such as aminoethyl acrylate and mono-2-(acryloxy)-ethylsuccinate; polymaleic acid; poly(acrylamides) such as polyacrylamide perse, poly(methacrylamide), poly(dimethylacrylamide), andpoly(N-isopropyl-acrylamide); poly(olefinic alcohol)s such as poly(vinylalcohol); poly(N-vinyl lactams) such as poly(vinyl pyrrolidone),poly(N-vinyl caprolactam), and copolymers thereof, polyoxazolines,including poly(methyloxazoline) and poly(ethyloxazoline); silicones,polyvinyl silicates, tetramethoxyorthosilicates,methyltrimethoxyorthosilicates, tetraalkoxyorthosilicates,trialkoxyorthosilicates, pressure sensitive silicone adhesives (such asBioPSA from Dow-Corning), and polyvinyl amines.

The gelling agent according to some embodiments of the presentdisclosure may include a polymer selected from any of synthetic orsemi-synthetic polymeric materials, polyacrylate copolymers, cellulosederivatives and polymethyl vinyl ether/maleic anhydride copolymers. Insome embodiments, the hydrophilic polymer comprises a polymer that is ahigh molecular weight (i.e., molar masses of more than about 5,000, andin some instances, more than about 10,000, or about 100,000, or about1,000,000) and/or cross-linked polyacrylic acid polymer.

In some embodiments, the gelling agent comprises a carbomer. Carbomersare synthetic high molecular weight polymer of acrylic acid that arecross-linked with either allylsucrose or allylethers of pentaerythritolhaving a molecular weight of about 3×10⁶. The gelation mechanism dependson neutralization of the carboxylic acid moiety to form a soluble salt.The polymer is hydrophilic and produces sparkling clear gels whenneutralized. Carbomer gels possess good thermal stability in that gelviscosity and yield value are essentially unaffected by temperature. Asa topical product, carbomer gels possess optimum rheological properties.The inherent pseudoplastic flow permits immediate recovery of viscositywhen shear is terminated and the high yield value and quick break makeit ideal for dispensing. Aqueous solution of Carbopol® is acidic innature due to the presence of free carboxylic acid residues.Neutralization of this solution cross-links and gelatinizes the polymerto form a viscous integral structure of desired viscosity.

Carbomers are available as fine white powders which disperse in water toform acidic colloidal suspensions (a 1% dispersion has a pH ofapproximately 3) of low viscosity. Neutralization of these suspensionsusing a base, for example sodium, potassium or ammonium hydroxides, lowmolecular weight amines and alkanolamines, results in the formation oftranslucent gels. Nicotine salts such as nicotine chloride form stablewater-soluble complexes with carbomers at about pH 3.5 and arestabilized at an optimal pH of about 5.6.

In some embodiments of the disclosure, the carbomer is Carbopol®. Suchpolymers are commercially available from B.F. Goodrich or Lubrizol underthe designation Carbopol® 71G NF, 420, 430, 475, 488, 493, 910, 934,934P, 940, 971PNF, 974P NF, 980 NF, 981 NF and the like. Carbopols areversatile controlled-release polymers, as described by Brock(Pharmacotherapy, 14:430-7 (1994), incorporated herein by reference) andDurrani (Pharmaceutical Res. (Supp.) 8:S-135 (1991), incorporated hereinby reference), and belong to a family of carbomers which are synthetic,high molecular weight, non-linear polymers of acrylic acid, crosslinkedwith polyalkenyl polyether. In some embodiments, the carbomer isCarbopol® 974P NF, 980 NF, 5984 EP, ETD 2020NF, Ultrez 10 NF, 934 NF,934P NF or 940 NF. In some embodiments, the carbomer is Carbopol® 980NF, ETD 2020 NF, Ultrez 10 NF, Ultrez 21 or 1382 Polymer, 1342 NF, 940NF. In some embodiments, about 0.05% to about 10%, about 0.5% to about5%, or about 1% to about 3% by weight of the total composition of a highmolecular weight carbopol can be present as the gelling agent. In someembodiments, the biophotonic composition of the disclosure comprisesfrom about 0.05% to about 10%, from about 0.5% to about 5%, or fromabout 1% to about 3% by weight of the total composition of a highmolecular weight carbopol.

In some embodiments, the gelling agent comprises a hygroscopic and/or ahydrophilic material useful for their water attracting properties. Thehygroscopic or hydrophilic material may include, but is not limited to,glucosamine, glucosamine sulfate, polysaccharides, cellulose derivatives(hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxypropylcellulose, methylcellulose and the like), noncellulose polysaccharides(galactomannans, guar gum, carob gum, gum arabic, sterculia gum, agar,alginates and the like), glycosaminoglycan, poly(vinyl alcohol),poly(2-hydroxyethylmethylacrylate), polyethylene oxide, collagen,chitosan, alginate, a poly(acrylonitrile)-based hydrogel, poly(ethyleneglycol)/poly(acrylic acid) interpenetrating polymer network hydrogel,polyethylene oxide-polybutylene terephthalate, hyaluronic acid,high-molecular-weight polyacrylic acid, poly(hydroxy ethylmethacrylate),poly(ethylene glycol), tetraethylene glycol diacrylate, polyethyleneglycol methacrylate, and poly(methyl acrylate-co-hydroxyethyl acrylate).In some embodiments, the hydrophilic gelling agent is selected fromglucose, modified starch, methyl cellulose, carboxymethyl cellulose,propyl cellulose, hydroxypropyl cellulose, carbomers, alginic acid,sodium alginate, potassium alginate, ammonium alginate, calciumalginate, agar, carrageenan, locust bean gum, pectin, and gelatin.

The gelling agent may be protein-based/naturally derived material suchas sodium hyaluronate, gelatin or collagen, lipids, or the like. Thegelling agent may be a polysaccharide such as starch, chitosan, chitin,agarose, agar, locust bean gum, carrageenan, gellan gum, pectin,alginate, xanthan, guar gum, and the like.

In some embodiments, the composition can include up to about 2% byweight of the final composition of sodium hyaluronate as the singlegelling agent. In some embodiments, the composition can include morethan about 4% or more than about 5% by weight of the final compositionof gelatin as the single gelling agent. In some embodiments, thecomposition can include up to about 10% or up to about 8% starch as thesingle gelling agent. In some embodiments, the composition can includemore than about 5% or more than about 10% by weight of the totalcomposition of collagen as the gelling agent. In some embodiments, about0.1% to about 10% or about 0.5% to about 3% by weight of the totalcomposition of chitin can be used as the gelling agent. In someembodiments, about 0.5% to about 5% by weight of the total compositionof corn starch or about 5% to about 10% by weight of the totalcomposition of corn starch can be used as the gelling agent. In someembodiments, more than about 2.5 wt % by weight of the total compositionof alginate can be used in the composition as the gelling agent. In someembodiments, the percentages by weight percent of the total compositionof the gelling agents can be as follows: cellulose gel (from about 0.3%to about 2.0%), konjac gum (from about 0.5% to about 0.7%), carrageenangum (from about 0.02% to about 2.0%), xanthan gum (from about 0.01% toabout 2.0%), acacia gum (from about 3% to about 30%), agar (from about0.04% to about 1.2%), guar gum (from about 0.1% to about 1%), locustbean gum (from about 0.15% to about 0.75%), pectin (from about 0.1% toabout 0.6%), tara gum (from about 0.1% to about 1.0%),polyvinylypyrrolidone (from about 1% to about 5%), sodium polyacrylate(from about 1% to about 10%). Other gelling agents can be used inamounts sufficient to gel the composition or to sufficiently thicken thecomposition. It will be appreciated that lower amounts of the abovegelling agents may be used in the presence of another gelling agent or athickener.

In some embodiments, the biophotonic composition of the presentdisclosure may be further encapsulated, e.g., in a membrane. Such amembrane may be transparent, and/or substantially, or fully impermeable.The membrane may be impermeable to liquid but permeable to gases such asair. In some embodiments, the composition may form a membrane thatencapsulates the chromophore(s) of the biophotonic topical composition,where the membrane may be substantially impermeable to liquid and/orgas. The membrane may be formed of one or more lipidic agents, polymers,gelatin, cellulose or cyclodextrins, or the like. In some embodiments,the membrane is translucent or transparent to allow light to infiltrateto and from the chromophore(s). In some embodiments, the composition isa dendrimer with an outer membrane comprising poly(propylene amine). Insome embodiments, the outer membrane comprises gelatin.

Polyols

According to some embodiments, the biophotonic compositions of thepresent disclosure may optionally further comprise one or more polyols.Suitable polyols that may be included in the composition include, butare not limited to a diol, a triol, a saccharide, glycerine,butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, propyleneglycol, butanediol, butenediol, butynediol, pentanediol, hexanediol,octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, diethyleneglycol, triethylene glycol, tetraethylene glycol, dipropylene glycol anddibutylene glycol. In some embodiments when the biophotonic compositionof the disclosure includes one or more polyols, the polyol is glycerine.In some embodiments when the biophotonic composition of the disclosureincludes one or more polyols, the polyol is propylene glycol. In someembodiments when the biophotonic composition of the disclosure includesone or more polyols, the polyol is a combination of glycerine andpropylene glycol.

In some embodiments, one or more polyols are present in an amount ofabout 5-75% by weight of the total composition, such as 5-75% by weightof the total composition. In some embodiments, one or more polyols arepresent in an amount of about 10-75% by weight of the total composition,such as 10-75% by weight of the total composition. In some embodiments,one or more polyols are present in an amount of about 15-75% by weightof the total composition, such as 15-75% by weight of the totalcomposition. In some embodiments, one or more polyols are present in anamount of about 20-75% by weight of the total composition, such as20-75% by weight of the total composition.

Antimicrobials

According to some embodiments, the biophotonic compositions of thepresent disclosure may optionally further comprise one or moreantimicrobials. Antimicrobials kill microbes or inhibit their growth oraccumulation. Exemplary antimicrobials (or antimicrobial agent) arerecited in U.S. Patent Application Publication Nos. 20040009227 and20110081530. Suitable antimicrobials for use in the methods of thepresent disclosure include, but not limited to, phenolic and chlorinatedphenolic and chlorinated phenolic compounds, resorcinol and itsderivatives, bisphenolic compounds, benzoic esters (parabens),halogenated carbonilides, polymeric antimicrobial agents, thazolines,trichloromethylthioimides, natural antimicrobial agents (also referredto as “natural essential oils”), metal salts, and broad-spectrumantibiotics.

Specific phenolic and chlorinated phenolic antimicrobial agents that canbe used in the disclosure include, but are not limited to: phenol;2-methyl phenol; 3-methyl phenol; 4-methyl phenol; 4-ethyl phenol;2,4-dimethyl phenol; 2,5-dimethyl phenol; 3,4-dimethyl phenol;2,6-dimethyl phenol; 4-n-propyl phenol; 4-n-butyl phenol; 4-n-amylphenol; 4-tert-amyl phenol; 4-n-hexyl phenol; 4-n-heptyl phenol; mono-and poly-alkyl and aromatic halophenols; p-chlorophenyl; methylp-chlorophenol; ethyl p-chlorophenol; n-propyl p-chlorophenol; n-butylp-chlorophenol; n-amyl p-chlorophenol; sec-amyl p-chlorophenol; n-hexylp-chlorophenol; cyclohexyl p-chlorophenol; n-heptyl p-chlorophenol;n-octyl; p-chlorophenol; o-chlorophenol; methyl o-chlorophenol; ethylo-chlorophenol; n-propyl o-chlorophenol; n-butyl o-chlorophenol; n-amylo-chlorophenol; tert-amyl o-chlorophenol; n-hexyl o-chlorophenol;n-heptyl o-chlorophenol; o-benzyl p-chlorophenol; o-benxyl-m-methylp-chlorophenol; o-benzyl-m,m-dimethyl, p-chlorophenol; o-phenylethylp-chlorophenol; o-phenylethyl-m-methyl p-chlorophenol; 3-methylp-chlorophenol 3,5-dimethyl p-chlorophenol, 6-ethyl-3-methylp-chlorophenol, 6-n-propyl-3-methyl p-chlorophenol;6-iso-propyl-3-methyl p-chlorophenol; 2-ethyl-3,5-dimethylp-chlorophenol; 6-sec-butyl-3-methyl p-chlorophenol;2-iso-propyl-3,5-dimethyl p-chlorophenol; 6-diethylmethyl-3-methylp-chlorophenol; 6-iso-propyl-2-ethyl-3-methyl p-chlorophenol;2-sec-amyl-3,5-dimethyl p-chlorophenol; 2-diethylmethyl-3,5-dimethylp-chlorophenol; 6-sec-octyl-3-methyl p-chlorophenol; p-chloro-m-cresolp-bromophenol; methyl p-bromophenol; ethyl p-bromophenol; n-propylp-bromophenol; n-butyl p-bromophenol; n-amyl p-bromophenol; sec-amylp-bromophenol; n-hexyl p-bromophenol; cyclohexyl p-bromophenol;o-bromophenol; tert-amyl o-bromophenol; n-hexyl o-bromophenol;n-propyl-m,m-dimethyl o-bromophenol; 2-phenyl phenol; 4-chloro-2-methylphenol; 4-chloro-3-methyl phenol; 4-chloro-3,5-dimethyl phenol;2,4-dichloro-3,5-dimethylphenol; 3,4,5,6-tetabromo-2-methylphenol;5-methyl-2-pentylphenol; 4-isopropyl-3-methylphenol;para-chloro-metaxylenol (PCMX); chlorothymol; phenoxyethanol;phenoxyisopropanol; and 5-chloro-2-hydroxydiphenylmethane.

Resorcinol and its derivatives can also be used as antimicrobial agents.Specific resorcinol derivatives include, but are not limited to: methylresorcinol; ethyl resorcinol; n-propyl resorcinol; n-butyl resorcinol;n-amyl resorcinol; n-hexyl resorcinol; n-heptyl resorcinol; n-octylresorcinol; n-nonyl resorcinol; phenyl resorcinol; benzyl resorcinol;phenylethyl resorcinol; phenylpropyl resorcinol; p-chlorobenzylresorcinol; 5-chloro-2,4-dihydroxydiphenyl methane;4′-chloro-2,4-dihydroxydiphenyl methane; 5-bromo-2,4-dihydroxydiphenylmethane; and 4′-bromo-2,4-dihydroxydiphenyl methane.

Specific bisphenolic antimicrobial agents that can be used in thedisclosure include, but are not limited to: 2,2′-methylenebis-(4-chlorophenol); 2,4,4′trichloro-2′-hydroxy-diphenyl ether, whichis sold by Ciba Geigy, Florham Park, N.J. under the trade nameTriclosan®; 2,2′-methylene bis-(3,4,6-trichlorophenol); 2,2′-methylenebis-(4-chloro-6-bromophenol);bis-(2-hydroxy-3,5-dichlorophenyl)sulphide; andbis-(2-hydroxy-5-chlorobenzyl)sulphide.

Specific benzoic esters (parabens) that can be used in the disclosureinclude, but are not limited to: methylparaben; propylparaben;butylparaben; ethylparaben; isopropylparaben; isobutylparaben;benzylparaben; sodium methylparaben; and sodium propylparaben.

Specific halogenated carbanilides that can be used in the disclosureinclude, but are not limited to: 3,4,4′-trichlorocarbanilides, such as3-(4-chlorophenyl)-1-(3,4-dichlorphenyl)urea sold under the tradenameTriclocarban® by Ciba-Geigy, Florham Park, N.J.;3-trifluoromethyl-4,4′-dichlorocarbanilide; and3,3′,4-trichlorocarbanilide.

Specific polymeric antimicrobial agents that can be used in thedisclosure include, but are not limited to: polyhexamethylene biguanidehydrochloride; and poly(iminoimidocarbonyl iminoimidocarbonyliminohexamethylene hydrochloride), which is sold under the tradenameVantocil® IB.

Specific thazolines that can be used in the disclosure include, but arenot limited to that sold under the tradename Micro-Check®; and2-n-octyl-4-isothiazolin-3-one, which is sold under the tradenameVinyzene® IT-3000 DIDP.

Specific trichloromethylthioimides that can be used in the disclosureinclude, but are not limited to: N-(trichloromethylthio)phthalimide,which is sold under the tradename Fungitrol®; andN-trichloromethylthio-4-cyclohexene-1,2-dicarboximide, which is soldunder the tradename Vancide®.

Specific natural antimicrobial agents that can be used in the disclosureinclude, but are not limited to, oils of: anise, lemon, orange,rosemary, wintergreen, thyme, lavender, cloves, hops, tea tree,citronella, wheat, barley, lemongrass, cedar leaf, cedarwood, cinnamon,fleagrass, geranium, sandalwood, violet, cranberry, eucalyptus, vervain,peppermint, gum benzoin, basil, honey, fennel, fir, balsam, menthol,ocmea origanuin, hydastis, carradensis, Berberidaceac daceae, Ratanhiaelonga, and Curcuma longa. Also included in this class of naturalantimicrobial agents are the key chemical components of the plant oilswhich have been found to provide antimicrobial benefit. These chemicalsinclude, but are not limited to: anethol, catechole, camphene, thymol,eugenol, eucalyptol, ferulic acid, farnesol, hinokitiol, tropolone,limonene, menthol, methyl salicylate, carvacol, terpineol, verbenone,berberine, ratanhiae extract, caryophellene oxide, citronellic acid,curcumin, nerolidol, and geraniol.

Specific metal salts that can be used in the disclosure include, but arenot limited to, salts of metals in Groups 3a-5a, 3b-7b, and 8 of theperiodic table. Specific examples of metal salts include, but are notlimited to, salts of: aluminum, zirconium, zinc, silver, gold, copper,lanthanum, tin, mercury, bismuth, selenium, strontium, scandium,yttrium, cerium, praseodymiun, neodymium, promethum, samarium, europium,gadolinium, terbium, dysprosium, holmium, erbium, thalium, ytterbium,lutetium, and mixtures thereof. An example of the metal-ion basedantimicrobial agent is sold under the tradename HealthShield®, and ismanufactured by HealthShield Technology, Wakefield, Mass.

Specific broad-spectrum antimicrobial agents that can be used in thedisclosure include, but are not limited to, those that are recited inother categories of antimicrobial agents herein.

Additional antimicrobial agents that can be used in the methods of thedisclosure include, but are not limited to: pyrithiones, and inparticular pyrithione-including zinc complexes such as these sold underthe tradename Octopirox®; dimethyidimethylol hydantoin, which is soldunder the tradename Glydant®;methylchloroisothiazolinone/methylisothiazolinone, which is sold underthe tradename Kathon CG®; sodium sulfite; sodium bisulfite;imidazolidinyl urea, which is sold under the tradename Germall 115®;diazolidinyl urea, which is sold under the tradename Germall 11®; benzylalcohol v2-bromo-2-nitropropane-1,3-diol, which is sold under thetradename Bronopol®; formalin or formaldehyde; iodopropenylbutylcarbamate, which is sold under the tradename Polyphase P100®;chloroacetamide; methanamine; methyldibromonitrile glutaronitrile(1,2-dibromo-2,4-dicyanobutane), which is sold under the tradenameTektamer®; glutaraldehyde; 5-bromo-5-nitro-1,3-dioxane, which is soldunder the tradename Bronidox®; phenethyl alcohol; o-phenylphenol/sodiumo-phenylphenol sodium hydroxymethylglycinate, which is sold under thetradename Suttocide A®; polymethoxy bicyclic oxazolidine; which is soldunder the tradename Nuosept C®; dimethoxane; thimersal; dichlorobenzylalcohol; captan; chlorphenenesin; dichlorophene; chlorbutanol; glyceryllaurate; halogenated diphenyl ethers;2,4,4′-trichloro-2′-hydroxy-diphenyl ether, which is sold under thetradename Triclosan® and is available from Ciba-Geigy, Florham Park,N.J.; and 2,2′-dihydroxy-5,5′-dibromo-diphenyl ether.

Additional antimicrobial agents that can be used in the methods of thedisclosure include those disclosed by U.S. Pat. Nos. 3,141,321;4,402,959; 4,430,381; 4,533,435; 4,625,026; 4,736,467; 4,855,139;5,069,907; 5,091,102; 5,639,464; 5,853,883; 5,854,147; 5,894,042; and5,919,554, and U.S. Pat. Appl. Publ. Nos. 20040009227 and 20110081530,the contents of all of which are incorporated herein by reference.

Additional Components

The compositions, methods, and uses of the disclosure can also includeother ingredients such as humectants (e.g., glycerine, ethylene glycol,and propylene glycol), preservatives such as parabens, and pH adjusterssuch as sodium hydroxide, sodium bicarbonate, and HCl.

In some embodiments, the pH of the composition is in or adjusted to therange of about 4 to about 10, such as from about 4 to about 9, fromabout 4 to about 8, from about 4 to about 7, from about 4 to 6.5, fromabout 4 to 6, from about 4 to 5.5, from 4 to 5. In some embodiments, thepH of the composition is in or adjusted to the range of from about 4 toabout 9. In some embodiments, the pH of the composition is in oradjusted to the range of from about 4 to about 8. In some embodiments,the pH of the composition is within the range of from about 4 to about7. In some embodiments, the pH of the composition is within the range ofabout from 4 to about 6.5. In some embodiments, the pH of thecomposition is within the range of from about 4 to about 6. In someembodiments, the pH of the composition is within the range of from about4 to about 5.5. In some embodiments, the pH of the composition is withinthe range of from about 4 to about 5. In some embodiments, the pH of thecomposition is within the range of about 5.0 to about 8.0, such as fromabout 6.0 to about 8.0, from about 6.5 to about 7.5, from about 5.5 toabout 7.5. In some embodiments, the pH of the composition is within therange of from about 6.0 to about 8.0. In some embodiments, the pH of thecomposition is within the range of from about 6.5 to about 7.5. In someembodiments, the pH of the composition is within the range of from about5.5 to about 7.5.

In some embodiments, the pH of the composition is in or adjusted to therange of from 4 to 10, such as from 4 to 9, from 4 to 8, from 4 to 7,from 4 to 6.5, from 4 to 6, from 4 to 5.5, from 4 to 5, from 5.0 to 8.0,from 6.0 to 8.0, from 6.5 to 7.5, from 5.5 to 7.5. In some embodiments,the pH of the composition is in or adjusted to the range of 4 to 9. Insome embodiments, the pH of the composition is in or adjusted to therange of 4 to 8. In some embodiments, the pH of the composition iswithin the range of 4 to 7. In some embodiments, the pH of thecomposition is within the range of 4 to 6.5. In some embodiments, the pHof the composition is within the range of 4 to 6. In some embodiments,the pH of the composition is within the range of 4 to 5.5. In someembodiments, the pH of the composition is within the range of 4 to 5. Insome embodiments, the pH of the composition is within the range of 5.0to 8.0. In some embodiments, the pH of the composition is within therange of 6.0 to 8.0. In some embodiments, the pH of the composition iswithin the range of 6.5 to 7.5. In some embodiments, the pH of thecomposition is within the range of 5.5 to 7.5.

In some embodiments, the biophotonic compositions of the disclosure alsoinclude an aqueous substance (such as water) or an alcohol. Alcoholsinclude, but are not limited to, ethanol, propanol, isopropanol,butanol, iso-butanol, t-butanol or pentanol. In some embodiments, thechromophore or combination of chromophores is in solution in a medium ofthe biophotonic composition. In some embodiments, the chromophore orcombination of chromophores is in solution in a medium of thebiophotonic composition, wherein the medium is an aqueous substance.

Methods of use and Treatment

Photoactivation

The biophotonic compositions suitable for use in the methods of thepresent disclosure may be selected from any of the embodiments of thebiophotonic compositions described above. For instance, the biophotoniccompositions useful in the method of the present disclosure may comprisea chromophore, such as a chromophore that undergoes at least partialphotobleaching upon application of light. The chromophore may absorb ata wavelength of from about 200 nm to about 800 nm, such as, from about200 nm to about 700 nm, from about 200 nm to about 600 nm or from about200 nm to about 500 nm. In some embodiments, the chromophore absorbs ata wavelength of from about 200 nm to about 600 nm. In some embodiments,the chromophore absorbs light at a wavelength of from about 200 nm toabout 300 nm, from about 250 nm to about 350 nm, from about 300 nm toabout 400 nm, from about 350 nm to about 450 nm, from about 400 nm toabout 500 nm, from about 450 nm to about 650 nm, from about 600 nm toabout 700 nm, from about 650 nm to about 750 nm or from about 700 nm toabout 800 nm. In some embodiments, suitable biophotonic compositions forthe methods of the present disclosure may further comprise at least oneadditional chromophore (e.g., a second chromophore). The absorptionspectrum of the second chromophore overlaps at least about 80%, about70%, about 60%, about 50%, about 40%, about 30%, or about 20% with theemission spectrum of the first chromophore. In some embodiments, thefirst chromophore has an emission spectrum that overlaps at least about1-10%, about 5-15%, about 10-20%, about 15-25%, about 20-30%, about25-35%, about 30-40%, about 35-45%, about 50-60%, about 55-65% or about60-70% with an absorption spectrum of the second chromophore.

Illumination of the biophotonic composition with light may cause atransfer of energy from the first chromophore to the second chromophore.Subsequently, the second chromophore may emit energy as fluorescenceand/or generate reactive oxygen species. In some embodiments of themethods the present disclosure, energy transfer caused by theapplication of light is not accompanied by concomitant generation ofheat, or does not result in tissue damage.

In the methods of the present disclosure, any source of actinic lightcan be used to illuminate the biophotonic compositions. Any type ofhalogen, LED or plasma arc lamp or laser may be suitable. The primarycharacteristic of suitable sources of actinic light will be that theyemit light in a wavelength (or wavelengths) appropriate for activatingthe one or more photoactivators present in the composition. In someembodiments, an argon laser is used. In some embodiments, apotassium-titanyl phosphate (KTP) laser (e.g., a GreenLight™ laser) isused. In another embodiment, sunlight may be used. In some embodiments,a LED photocuring device is the source of the actinic light. In someembodiments, the source of the actinic light is a source of light havinga wavelength from about 200 nm to about 800 nm. In some embodiments, thesource of the actinic light is a source of visible light having awavelength between about 400 nm and about 700 nm. In some embodiments,the source of the actinic light is a source of visible light having awavelength between about 400 nm and about 600 nm. In some embodiments,the source of the actinic light is a source of visible light having awavelength between about 400 nm and about 550 nm. In some embodiments,the source of the actinic light is a source of visible light having awavelength between about 380 nm and about 700 nm. In some embodiments,the source of the actinic light is a source of visible light having awavelength between about 380 nm and about 600 nm. In some embodiments,the source of the actinic light is a source of visible light having awavelength between about 380 nm and about 550 nm. In some embodiments,the source of the actinic light is a source of light having a wavelengthbetween 200 nm to 800 nm. In some embodiments, the source of the actiniclight is a source of visible light having a wavelength between 400 nmand 700 nm. In some embodiments, the source of the actinic light is asource of visible light having a wavelength between 400 nm and 600 nm.In some embodiments, the source of the actinic light is a source ofvisible light having a wavelength between 400 nm and 550 nm. In someembodiments, the source of the actinic light is a source of visiblelight having a wavelength between 380 nm and 700 nm. In someembodiments, the source of the actinic light is a source of visiblelight having a wavelength between 380 nm and 600 nm. In someembodiments, the source of the actinic light is a source of visiblelight having a wavelength between 380 nm and 550 nm. In someembodiments, the biophotonic composition of the disclosure isilluminated with violet and/or blue light. Furthermore, the source ofactinic light should have a suitable power density. Suitable powerdensity for non-collimated light sources (LED, halogen or plasma lamps)are in the range from about 1 mW/cm² to about 1200 mW/cm², such as fromabout 50 mW/cm² to about 1000 mW/cm²′ from 100 mW/cm² to about 900mW/cm², from 200 mW/cm² to about 800 mW/cm², or from about 1 mW/cm² toabout 200 mW/cm². In some embodiments, suitable power density fornon-collimated light sources (LED, halogen or plasma lamps) are in therange from about 1 mW/cm² to about 200 mW/cm². In some embodiments,suitable power density for laser light sources is in the range fromabout 0.5 mW/cm² to about 0.8 mW/cm².

In some embodiments of the methods of the present disclosure, the lighthas an energy at the patient's skin of from about 1 mW/cm² to about 500mW/cm², or about 1-300 mW/cm², or about 1-200 mW/cm², wherein the energyapplied depends at least on the condition being treated, the wavelengthof the light, the distance of the patient's skin from the light source,and the thickness of the biophotonic composition. In some embodiments,the light at the patient's skin is from about 1 to about 40 mW/cm², orabout 20-60 mW/cm², or about 40-80 mW/cm², or about 60-100 mW/cm², orabout 80-120 mW/cm², or about 100-140 mW/cm², or about 120-160 mW/cm²,or about 140-180 mW/cm², or about 160-200 mW/cm², or about 110-240mW/cm², or about 110-150 mW/cm², or about 190-240 mW/cm².

In some embodiments, a mobile device can be used to activate embodimentsof the biophotonic composition of the present disclosure, wherein themobile device can emit light having an emission spectrum which overlapsan absorption spectrum of the chromophore in the biophotoniccomposition. The mobile device can have a display screen through whichthe light is emitted and/or the mobile device can emit light from aflashlight which photoactivates the biophotonic composition.

In some embodiments, a display screen on a television or a computermonitor can be used to activate the biophotonic composition, wherein thedisplay screen can emit light having an emission spectrum which overlapsan absorption spectrum of a photoactive agent in the photoactivatablecomposition.

In some embodiments, the chromophore or combination of chromophores canbe photoactivated by ambient light which may originate from the sun orother light sources. Ambient light can be considered to be a generalillumination that comes from all directions in a room that has novisible source. In some embodiments, the chromophore or combination ofchromophores can be photoactivated by light in the visible range of theelectromagnetic spectrum. Exposure times to ambient light may be longerthan that to direct light.

In some embodiments, different sources of light can be used to activatethe biophotonic compositions, such as a combination of ambient light anddirect LED light.

The duration of the exposure to actinic light required will be dependenton the surface of the treated area, the severity of the condition thatis being treated, the power density, wavelength and bandwidth of thelight source, the thickness of the biophotonic composition, and thetreatment distance from the light source. The illumination of thetreated area by fluorescence may take place within seconds or evenfragment of seconds, but a prolonged exposure period is beneficial toexploit the synergistic effects of the absorbed, reflected and reemittedlight on the composition of the present disclosure and its interactionwith the tissue being treated. In some embodiments, the time of exposureto actinic light of the tissue or skin or wound on which the biophotoniccomposition has been applied is a period from about 1 second to about 60minutes. In some embodiments, the time of exposure to actinic light ofthe tissue or skin or wound on which the biophotonic composition hasbeen applied is a period from about 1 minute to about 60 minutes. Insome embodiments, the time of exposure to actinic light of the tissue,skin or wound on which the biophotonic composition has been applied is aperiod from about 1 minute to about 5 minutes. In some embodiments, thetime of exposure to actinic light of the tissue, skin or wound on whichthe biophotonic composition has been applied is a period from about 1minute and 5 minutes. In another embodiment, the time of exposure isfrom about 1 second to about 5 minutes or from about 60 seconds to about5 minutes. In another embodiment, the time of exposure to actinic lightof the tissue on which the biophotonic composition has been applied is aperiod of less than about 5 minutes. In another embodiment, the time ofexposure is from about 1 second to about 5 minutes, or from about 60seconds and about 5 minutes per cm² of the area to be treated, so thatthe total time of exposure of a 10 cm² area would be from 10 minutes and50 minutes.

In some embodiments, the biophotonic composition is illuminated for aperiod from about 1 minute to about 3 minutes. In some embodiments,light is applied for a period from about lto about 30 seconds, fromabout 1 to about 60 seconds, from about 15 seconds to about 45 seconds,from about 30 seconds to about 60 seconds, from about 0.75 minute toabout 1.5 minutes, from about 1 minute to about 2 minutes, from about1.5 minute to about 2.5 minutes, from about 2 minutes to about 3minutes, from about 2.5 minutes to about 3.5 minutes, from about 3minutes to about 4 minutes, from about 3.5 minutes to about 4.5 minutes,from about 4 minutes to about 5 minutes, from about 5 minutes to about10 minutes, from about 10 minutes to about 15 minutes, from about 15minutes to about 20 minutes, from about 20 minutes to about 25 minutes,or from about 20 minutes to about 30 minutes. In some embodiments, lightis applied for a period of about 1 second. In some embodiments, light isapplied for a period of about 5 seconds. In some embodiments, light isapplied for a period of about 10 seconds. In some embodiments, light isapplied for a period of about 20 seconds. In some embodiments, light isapplied for a period of about 30 seconds. In some embodiments, thebiophotonic composition is illuminated for a period less than about 60minutes. In some embodiments, the biophotonic composition is illuminatedfor a period less than about 30 minutes. In some embodiments, thebiophotonic composition is illuminated for a period less than about 20minutes. In some embodiments, the biophotonic composition is illuminatedfor a period less than about 15 minutes. In some embodiments, thebiophotonic composition is illuminated for a period less than about 10minutes. In some embodiments, the biophotonic composition is illuminatedfor a period less than about 5 minutes. In some embodiments, thebiophotonic composition is illuminated for a period less than about 1minute. In some embodiments, the biophotonic composition is illuminatedfor a period less than about 30 seconds. In some embodiments, thebiophotonic composition is illuminated for a period less than about 20seconds. In some embodiments, the biophotonic composition is illuminatedfor a period less than about 10 seconds. In some embodiments, thebiophotonic composition is illuminated for a period less than about 5seconds. In some embodiments, the biophotonic composition is illuminatedfor a period less than about 1 second. In some embodiments, the sourceof actinic light is in continuous motion over the treated area for theappropriate time of exposure. In some embodiments, multiple applicationsof the biophotonic composition and actinic light are performed. In someembodiments, the tissue, skin or wound is exposed to actinic light atleast two, three, four, five or six times. In some embodiments, thetissue, skin or wound is exposed to actinic light at least two, three,four, five or six times with a resting period in between each exposure.In certain such embodiments, the resting period is less than about 1minute, less than about 5 minutes, less than about 10 minutes, less thanabout 20 minutes, less about 40 minutes, less than about 60 minutes,less than about 2 hours, less than about 4 hours, less than about 6hours, or less than 12 hours. In some embodiments, the entire treatmentmay be repeated in its entirety as may be required by the patient. Insome embodiments, a fresh application of the biophotonic composition isapplied before another exposure to actinic light.

In the methods of the present disclosure, the biophotonic compositionmay be optionally removed from the site of treatment followingapplication of light. In some embodiments, the biophotonic compositionis left on the treatment site for more than about 30 minutes, more thanone hour, more than about 2 hours, more than about 3 hours. It can beilluminated with ambient light. To prevent drying, the composition canbe covered with a transparent or translucent cover such as a polymerfilm, or an opaque cover which can be removed before illumination.

For any of the methods described herein, the embodiments of thisdisclosure contemplate the use of any of the compositions, or mixturesof them, described throughout the application. In addition, in variousembodiments of any of the methods described herein, combinations of anystep or steps of one method with any step or steps from another methodmay be employed.

Otitis Externa

The biophotonic compositions and methods of the present disclosure areuseful to treat otitis externa. Therefore, it is an objective of thepresent disclosure to provide a method of providing biophotonic therapyto a target site, wherein the method is for the treatment of otitisexterna. In certain embodiments, the otitis externa is chronic orrelapsing otitis externa.

Otitis externa is an inflammation or infection of the external auditorycanal, the auricle (pinna), or both. Causes of otitis extema in mammalsinclude, but are not limited to, allergies, such as atopy or foodallergies; parasites, such as ear mites; microorganisms, such asbacteria and yeast or other fungi; foreign bodies; trauma; the earenvironment, e.g., excess moisture and ear anatomy; hereditary or immuneconditions; and tumors. The condition is often chronic and one of themain aspects involved in the chronicity of the disease is the presenceof infections caused by bacteria resistant to different antibiotics(e.g., Pseudomonas aeruginosa).

Otitis externa is one of the most common conditions seen in cats anddogs. Symptoms of otitis externa in cats and dogs include odor,scratching or rubbing of ears and head, discharge in the ears, rednessor swelling of the ear canal, shaking of the head or tilting it to oneside, pain around the ears, and changes in behavior such as depressionor irritability.

Otitis externa is also a common condition in humans. Symptoms of otitisexterna in humans include otalgia, hearing loss, ear pressure, narrowingof the ear canal, tinnitus, fever, itching, pain, and discharge.

In some aspects, the disclosure provides a method of treating otitisexterna comprising: applying a biophotonic composition to a patient inneed thereof, wherein the biophotonic composition comprises at least oneoxidant and at least one chromophore capable of activating the oxidant;and exposing said biophotonic composition to actinic light for a timesufficient for said chromophore to cause activation of said oxidant. Incertain such aspects, the patient is a mammal, such as a human, a felineor a canine. In certain such aspects, the otitis externa is chronicotitis externa. In certain such aspects, the method is performed onceper week for one or more weeks, such as once per week for one week, twoweeks, three weeks, four weeks, five weeks, or six weeks, or up to whatis deemed appropriate by the physician or veterinarian. In certain suchaspects, the method is performed twice per week for one or more weeks,such as twice per week for one week, two weeks, three weeks, four weeks,five weeks, or six weeks, or up to what is deemed appropriate by thephysician or veterinarian.

In other aspects, the disclosure provides for the use of a biophotoniccomposition for the manufacture of a medicament for treating a patientafflicted with otitis externa, wherein said composition comprises: atleast one oxidant, and at least one chromophore capable of activatingthe oxidant; in association with a pharmacologically acceptable carrier.In certain such aspects, the patient is a mammal, such as a human, afeline or a canine. In certain such aspects, the otitis externa ischronic otitis externa.

In some aspects, the disclosure provides for use of a biophotoniccomposition for the treatment of a patient afflicted with otitisexterna, wherein said composition comprises: at least one oxidant; andat least one chromophore capable of activating the oxidant; inassociation with a pharmacologically acceptable carrier. In certain suchaspects, the patient is a mammal, such as a human, a feline or a canine.In certain such aspects, the otitis externa is chronic otitis externa.

In some embodiments, the biophotonic compositions of the disclosure maybe applied at regular intervals such as one or more times per week,and/or at an interval deemed appropriate by the physician orveterinarian. In some embodiments, the biophotonic compositions of thedisclosure are applied once per week for one or more weeks, such as onceper week for one week. In some embodiments, the biophotonic compositionsof the disclosure are applied once per week for two weeks. In someembodiments, the biophotonic compositions of the disclosure are appliedonce per week for three weeks. In some embodiments, the biophotoniccompositions of the disclosure are applied once per week for four weeks.In some embodiments, the biophotonic compositions of the disclosure areapplied once per week for five weeks. In some embodiments, thebiophotonic compositions of the disclosure are applied once per week forsix weeks. In some embodiments, the biophotonic compositions of thedisclosure are applied once per week for seven weeks. In someembodiments, the biophotonic compositions of the disclosure are appliedonce per week for eight or more weeks.

In some embodiments, the biophotonic compositions of the disclosure areapplied twice per week for one or more weeks, such as twice per week forone week. In some embodiments, the biophotonic compositions of thedisclosure are applied twice per week for two weeks. In someembodiments, the biophotonic compositions of the disclosure are appliedtwice per week for three weeks. In some embodiments, the biophotoniccompositions of the disclosure are applied twice per week for fourweeks. In some embodiments, the biophotonic compositions of thedisclosure are applied twice per week for five weeks. In someembodiments, the biophotonic compositions of the disclosure are appliedtwice per week for six weeks. In some embodiments, the biophotoniccompositions of the disclosure are applied twice per week for sevenweeks. In some embodiments, the biophotonic compositions of thedisclosure are applied twice per week for eight or more weeks.

In some embodiments, the biophotonic compositions of the disclosure areapplied three times or more per week for one or more weeks, such asthree times or more per week for one week, three times or more per weekfor two weeks. In some embodiments, the biophotonic compositions of thedisclosure are applied three times or more per week for three weeks. Insome embodiments, the biophotonic compositions of the disclosure areapplied three times or more per week for four weeks. In someembodiments, the biophotonic compositions of the disclosure are appliedthree times or more per week for five weeks. In some embodiments, thebiophotonic compositions of the disclosure are applied three times ormore per week for six weeks. In some embodiments, the biophotoniccompositions of the disclosure are applied three times or more per weekfor seven weeks. In some embodiments, the biophotonic compositions ofthe disclosure are applied three times or more per week for eight ormore weeks.

In some embodiments, the biophotonic compositions and methods of thepresent disclosure are useful in treating otitis externa, for example,by ameliorating any symptom caused by a microorganism or inhibiting itfrom spreading. In some embodiments, the biophotonic compositions andmethods of the present disclosure are useful in treating otitis externa,for example, by treating or preventing redness and swelling. In someembodiments, the biophotonic compositions and methods of the presentdisclosure are useful in treating otitis externa, for example, bytreating or preventing discharge from the ear. In some embodiments, thebiophotonic compositions and methods of the present disclosure areuseful in treating otitis externa without the use of antibiotics.

Combination Therapies

Any of the biophotonic compositions, methods, or uses of this disclosuremay be useful in combination with other therapeutics.

In some embodiments, the phrase “combination therapy” embraces theadministration of any of the compositions described herein, and anadditional therapeutic agent, or mixtures of them, as part of a specifictreatment regimen intended to provide a beneficial effect from theco-action of these therapeutic agents. Administration of thesetherapeutic agents in combination typically is carried out over adefined time period (usually minutes, hours, days or weeks dependingupon the combination selected). “Combination therapy” is intended toembrace administration of these therapeutic agents in a sequentialmanner, that is, wherein each therapeutic agent is administered at adifferent time, as well as administration of these therapeutic agents,or at least two of the therapeutic agents, in a substantiallysimultaneous manner. The therapeutic agents can be administered by thesame route or by different routes. For example, a first therapeuticagent of the combination selected may be administered by intravenousinjection or orally while the biophotonic composition of the disclosureis administered topically. Alternatively, for example, all therapeuticagents may be administered topically. The sequence in which thetherapeutic agents are administered is not narrowly critical.“Combination therapy” also embraces the administration of thecompositions as described herein in further combination with otherbiologically active ingredients (such as, but not limited to, a secondand different therapeutic agent) and/or non-drug therapies (such as, butnot limited to, surgery or radiation).

In some embodiments, the therapeutic agents administered in combinationtherapy simultaneously, separately, or sequentially with any of thecompounds and compositions of this disclosure, or mixtures thereof, cancomprise, but are not limited to: a non-steroidal anti-inflammatory drug(NSAID), an anti-inflammatory agent, a corticosteroid, an anti-allergicagent, a steroid drug, one or more of the antimicrobial agents describedabove, or mixtures thereof.

In some embodiments, any of the compositions described herein can allowthe combination therapeutic agents and/or compositions described hereinor mixtures thereof to be administered at a low dose, that is, at a doselower than has been conventionally used in clinical situations.

Alternatively, the methods and combinations of this disclosure maximizethe therapeutic effect at higher doses.

In some embodiments, when administered as a combination, the therapeuticagents can be formulated as separate compositions which are given at thesame time or different times, or the therapeutic agents can be given asa single composition.

Kits

The present disclosure also provides kits for preparing and/or applyingany of the compositions of the present disclosure for the treatment ofotitis externa. The kit may include a biophotonic topical composition asdescribed herein, a device for applying or removing the composition,instructions of use for the composition, and/or a light source. In someembodiments, the biophotonic composition comprises at least one oxidantand at least one chromophore capable of activating the oxidant.

In some embodiments, the kit includes more than one composition, forexample, a first and a second composition. The first composition mayinclude at least one chromophore capable of activating the oxidant andthe second composition may include at least one oxidant. In certain suchembodiments, the oxidant is chosen from hydrogen peroxide, carbamideperoxide and benzoyl peroxide. In certain such embodiments, the firstand/or second composition further comprises one or more gelling agents.

In some embodiments, the first composition may comprise at least onechromophore capable of activating the oxidant in a liquid or as apowder, and the second composition may comprise at least one oxidant. Incertain such embodiments, the oxidant is chosen from hydrogen peroxide,carbamide peroxide and benzoyl peroxide. In certain such embodiments,the first and/or second composition further comprises one or moregelling agents.

In some embodiments, the kit includes containers comprising thecompositions of the present disclosure. In some embodiments, the kitincludes a first container comprising the at least one chromophorecapable of activating the oxidant, and a second container comprising atleast one oxidant. In certain such embodiments, the oxidant is chosenfrom hydrogen peroxide, carbamide peroxide and benzoyl peroxide. Incertain such embodiments, the first and/or second composition furthercomprises one or more gelling agents.

The containers may be light impermeable, air-tight, and/or leakresistant. Exemplary containers include, but are not limited to,syringes, vials, or pouches. The first and second compositions may beincluded within the same container but separated from one another untila user mixes the compositions. For example, in certain such embodiments,the container may be a dual-chamber syringe where the contents of thechambers mix on expulsion of the compositions from the chambers. In someembodiments, the pouch may include two chambers separated by a frangiblemembrane. In some embodiments, one component may be contained in asyringe and injectable into a container comprising the second component.

The biophotonic composition may also be provided in a containercomprising one or more chambers for holding one or more components ofthe biophotonic composition, and an outlet in communication with the oneor more chambers for discharging the biophotonic composition from thecontainer.

In some embodiments, the kit comprises a systemic or topical drug foraugmenting the treatment of the composition. In certain suchembodiments, the kit may include a systemic or topical antibiotic orhormone treatment for otitis externa.

Written instructions on how to use the biophotonic composition inaccordance with the present disclosure may be included in the kit, ormay be included on or associated with the containers comprising thecompositions of the present disclosure.

In some embodiments, the kit may comprise a further component which is adressing. The dressing may be a porous or semi-porous structure forreceiving the biophotonic composition. The dressing may comprise wovenor non-woven fibrous materials.

In some embodiments of the kit, the kit may further comprise a lightsource such as a portable light with a wavelength appropriate toactivate the chromophore in the biophotonic composition. The portablelight may be battery operated or re-chargeable.

In some embodiments, the kit may further comprise one or morewaveguides.

Identification of equivalent compositions, methods and kits are wellwithin the skill of the ordinary practitioner and would require no morethan routine experimentation, in light of the teachings of the presentdisclosure. Practice of the disclosure will be still more fullyunderstood from the following examples, which are presented herein forillustration only and should not be construed as limiting the disclosurein any way.

EXAMPLES

The examples below are given so as to illustrate the practice of variousembodiments of the present disclosure. They are not intended to limit ordefine the entire scope of this disclosure.

It should be appreciated that the disclosure is not limited to theparticular embodiments described and illustrated herein but includes allmodifications and variations falling within the scope of the disclosureas defined in the appended embodiments.

Evaluation of Treatment with Biophotonic Therapy in Canines and Felines

Studies were carried out to evaluate the efficacy and effectiveness ofthe biophotonic compositions of the present description in treatment ofcanine and feline chronic otitis externa in comparison to conventionaltherapy. These studies provided information regarding resistantbacterial complications in chronic or relapsing otitis externa, which isone of the most important causes of chronicity of the disease. In thesestudies, factors that contribute to the development of the biophotonictherapy in otitis externa were assessed. These factors included but werenot limited to: viscosity, concentration, method of light-delivery,duration of radiation, and frequency of treatment. Results of treatmentin these studies offered resolution of chronic or relapsing otitisexterna bacterial complications.

Study Design

A randomized controlled clinical trial was conducted in dogs withclinically apparent, spontaneous, chronic and/or relapsed otitis externawith or without bacterial complications. After the integrity of thetympanic membrane was evaluated, dogs were divided into three groups:Group I (or Group QW)—treatment with biophotonic therapy once a week forsix times (T0 to T5) and evaluated after each treatment; Group II (orGroup BW)—treatment with biophotonic therapy twice a week for six times(T0-T5) and evaluated after each treatment; and Group III (or GroupC)—treatment with a conventional therapy twice-a-day for two weeks andtwice-a-week evaluation (T0 to T5). The biophotonic therapy was carriedout with no concomitant antibiotic and anti-inflammatory therapy. Theconventional therapy was a topical therapy with Baytril Otic® (emulsioncontaining enrofloxacin (5 mg/mL) and silver sulfadiazine (10 mg/mL)with benzyl alcohol (20 mg/mL), cetylstearyl alcohol in a neutral oiland purified water emulsion). In all three groups, before the firsttreatment, the external auditory channel was adequately cleaned. Earswabs for bacterial culture and cytology were sampled prior to thetreatment and once a week during the full length of the treatment.

Assessed Parameters and Tests

Clinical assessment included: (1) otitis index scoring system (OTIS-3)based on erythema, oedema/swelling, erosion/ulceratin, and exudate. Thetotal score was between 0-12 (Nattal & Benisgnor, 2014); (2) Pruritusseverity scale (VAS 0-10) (Hill et al., 2007, Rybnicek, et al., 2008;Hill et al., 2009); (3) Pain severity score (VAS 0-10) (Buback et al.,1996; Wolfe et al., 2006; Nutal & Bensignor, 2014); and (4) Actualtemperatures (° C.) taken before and/or after treatment in exposed andcontralateral ear canal (Grono, 1970; Cole, 2009; Mittal 2014).Preferably, temperatures may be taken before each application of thetreatment with liquids (e.g., saline) or the biophotonic composition.

Cytological assessment included: (1) visual description of thecytological findings; and (2) otitis cytological scoring system (0-3)for presence of cells, earwax/cerumen, neutrophils, bacteria (e.g., rodshaped bacteria, coccoid bacteria), fungi, or yeast, etc.

Bacteriologic assessment included: (1) bacterial culture; (2)quantitative bacteriological assessment (measured by colony formingunit, CFU) for total bacterial counts and bacterial counts for eachspecies in case of multiple isolations; and (3) antibioticsusceptibility testing.

General Protocols for Each Treatment Session

(1) Before treatment, perform ear swab for bacteriology assessment (FIG.4)

(2) Perform Ear swab for cytological assessment (FIG. 5)

(3) Before treatment, measure aural temperature bilaterally (FIG. 6)

(4) Before treatment, take photographs of the external meatus (FIG. 7)

(5) Record otoscopy and attribution clinical score

(6) Prepare therapeutic composition by mixing chromophore and ureaperoxide (UP) (FIG. 8A); apply the therapeutic composition locally (FIG.8B); illuminate the treated area with a Bluephase® lamp (IvoclarVivadent AG, FL-9494 Schaan, Liechtenstein), a source of actinic light(FIG. 9)

(7) Take an ear swab for bacteriologic assessment after treatment (FIG.10)

(8) Measure aural temperature bilaterally after treatment (FIG. 11)

(9) Take a photograph of the external meatus after treatment (FIG. 12)

Results

Forty three (43) patients with otitis were recruited and divided intothree groups: fifteen (15) cases in Group I (Group QW) and fourteen (14)cases in Group II (Group BW), and fourteen cases in Group III (Group C).The numbers of tests performed during the treatment totaled: 258clinical assessments, 492 aural temperature assessments, 258 non-sterileear swabs, 258 cytological assessments, 432 sterile ear swabs, 432bacteriologic assessments (including cultures, identification, bacterialcounts, and antibiotic susceptibility tests).

Treatments were provided with specifications according to the Table 1below in order to establish a therapeutic protocol with respect to theviscosity of composition to be applied into the ear canal of the animalto be treated and to establish a suitable regimen with respect to anillumination of the applied composition once present in the ear canal tobe treated.

TABLE 1 Test protocols for composition application for a treatment ofcanine otitis externa Bluephase lamp Formulation and Illumination lightemission Chromophore % UP^((a)) Light Source^((b)) Time program^((c))Liquid, Eosin Y 3 Blue phase lamp 10 sec Soft Start Liquid, Eosin Y 6Blue phase lamp 30 sec Soft Start Semi-liquid, 6 Blue phase lamp with  4min High Eosin Y multi-fiber Semi-liquid, 6 Blue phase lamp without 2-3min (in Soft Eosin Y multi-fiber some Start + High^((d)) instances, 1.5min or 30 sec)

With reference to Table 1 above, the symbol “(a)” stands for ureaperoxide (UP, carbamide peroxide) and % UP is the percent of UP byweight of the total composition. Symbol “(b)” denotes the use ofphotonic conductor, single-fiber or multi-fiber, to allow for a betterdistribution of light in the ear. It was found that it was not necessaryto use a single-fiber or a coated multi-fiber photonic conductor for thebiophotonic therapy. Symbol “(c)” indicates that multiple cycles wereperformed to achieve total illumination from 2 to 4 minutes as themaximum duration of emission of the light source was 30 seconds. Symbol“(d)” indicates the Bluephase® lamp illumination program that wasutilized: Soft Start program in the first cycle, then High program insubsequent cycles. The Soft Start and High programs for the Bluephase®lamp are pre-set illumination programs that are programmed into the lampby the manufacture and can be selected for use by following themanufacturer's instructions for use. For the Soft Start program, thelamp will proceed to increase the intensity of light in a step-by-stepfashion, e.g., from about 650 mW/cm² to about 1200 mW/cm², whereas withthe High program, the lamp will emit light at a consistently high level(e.g., approximately 1200 mW/cm²) upon initiation of the illuminationcycle.

Statistical analysis of data were carried our using Mann-Whitney ranksum test and Wilcoxon rank sum test for ordinal variables; Studentt-test for cardinal variables. A difference with a p-value <=0.05 willbe considered statistically significant.

An application of a composition of the present description in liquidform comprised a chromophore gel comprising a chromophore, a gellingagent (e.g., carbopol), and water; and a carrier gel comprising UP andwater.

The semi-liquid form of the composition comprised a carrier gel and achromophore gel. The carrier gel comprised UP, carbopol, and water andthe chromophore gel comprised a chromophore, a gelling agent and water.Carbopol was present in an amount of 0.6% (on a % w/w basis of the totalcomposition). The carrier gel and chromophore gel components of thecomposition had respective pH values of 5.24 and 5.09. The resultingbiophotonic compositions for application to the patient's ear canal hada semi-liquid consistency due to the low carbopol concentration and lowpH, thus, allowing for the composition to flow into the ear canal.

With respect to selecting a suitable protocol from those described inTable 1, the semi-liquid form of the composition was selected (see rows4 and 5, Table 1) with a total illumination time of 2 to 4 minutes. Theillumination program was:

1) A first 30 second illumination cycle using the Soft Start program,and

2) All subsequent illumination cycles using the High program.

For most treatment cases, a photonic conductor (light conducting) tipwas attached to the illumination end of the lamp to achieve a greaterdepth of illumination within the ear canal and, thus, the appliedcomposition.

(1) Results According to Clinical Score

Results relating to a clinical score with respect to the canine patientstreated with the biophotonic compositions of the present disclosureindicated that the treatment yielded positive improvement in the otitiscondition of the patients treated with the biophotonic compositions ofthe present disclosure. As can be seen from FIG. 13A, the overallerythema (redness of the skin in the ear canal) score decreased in thetreated animals, with a steep decline in the degree of redness occurringafter the initial treatment. Regarding oedema/swelling and exudate (fromthe ear canal), both of these factors also decreased (see FIGS. 13B and13D).

FIGS. 20A-20C present the data for all three Groups. In FIG. 20A, theoverall erythema (redness of the skin in the ear canal) score decreasedin patients of both Groups I (QW) and II (BW). For patients in Group I(QW), there was a steep decline in the degree of redness occurring afterthe initial treatment. Regarding the criteria of oedema/swelling,erosion/ulceration, and exudate (from the ear canal), all of thesefactors also showed a decrease in patients of both Group I (QW) and II(BW) (see FIGS. 20B, 20C, and 20D).

Combining the aforementioned four criteria to derive a total OtitisIndex Score (OTIS-3) (See, Nuttall, T. and Bensignor E. “A pilot studyto develop an objective clinical score for canine otitis externa,”Veterinary Dermatology, 2014, volume 25(6), pages 530-e92, incorporatedherein by reference), as can be seen in FIG. 14, indicates there was asteep decline in the score index up to the completion of the thirdtreatment, with the decrease maintained after completion of the fifthtreatment. The OTIS-3 score observed at the commencement of thetreatment, these data indicate a marked improvement in the treatedpatients' otitis condition from a clinical criteria perspective. FIG. 21indicates OTIS-3 scores for the three Groups. As one can see, a steepdecline was observed in the score index in both Group I (QW) and GroupII (BW). These data indicate a marked improvement in the treatedpatients' otitis condition from a clinical criteria perspective.

Data from other clinical criteria, such as an evaluation of pruritus(itchiness) using a pruritus severity scale (VAS 0-10) (see Hill P. B.et al. “Development of an owner-assessed scale to measure the severityof pruritus in dogs,” Veterinary Dermatology, 2007, volume 18(5), pages301-308; Rybníěek, J. et al. “Further validation of a pruritus severityscale for use in dogs,” Veterinary Dermatology, 2008, volume 20, pages115-122, each reference incorporated herein by reference) and a painseverity score (VAS 0-10) (see Nuttall, T. and Bensignor E. 104,referenced above), were also evaluated, see FIG. 15A and FIG. 15B,respectively, and these criteria also showed an improvement over thecourse of the otitis treatment via a biophotonic therapy using thebiophotonic compositions of the present description. In FIGS. 22 A and22B, pruritus and pain data are presented for all three Group I (QW),Group II (BW), and Group III (C).

A further clinical evaluation was also measured, aural temperatures inthe exposed and contralateral ear canals of patients both before andafter treatment with the biophotonic compositions of the disclosure ortreatment with a conventional therapy (see Grono L. R. “Studies of themicroclimate of the external auditory canal in the dog. I. AuralTemperature,” Research in Veterinary Sciences, 1970, volume 11(4), pages307-311, incorporated herein by reference). The results from the dogstreated with the biophotonic compositions of the present disclosure areshown in FIG. 16A. The results from dogs treated with conventionaltherapy are shown in FIG. 16B. The post-treatment temperature for dogsreceiving the biophotonic composition treatment showed a greaterdifferential increase to the pre-treatment temperature versus theconventional therapy treated group, as illustrated in FIG. 16C.

(2) Results According to Cytological Scoring System

Results with respect to cytological scoring indicia, which included aneutrophil count score and an earwax/cerumen measurement score, alongwith a relative score measurement of rod shaped bacteria, coccoidbacteria and malassezia (to measure the presence of yeast in the earcanal), indicated that animals afflicted with otitis externa and treatedwith the biophotonic compositions of the present description showed animprovement in these indicia.

As can be seen in FIG. 17A, the neutrophil score for treated dogsgradually decreased from the first to third treatments, and thereafterincreased after the fourth treatment, which may indicate that the immunesystems of the treated dogs had started to fight any bacterial andfungal infections related to the otitis condition. FIG. 23A presents theneutrophil score for all three Groups.

Regarding the presence of earwax/cerumen in the ear canals of the dogsthat received the biophotonic composition treatment regimen, as shown inFIG. 17B, there was an increase in the presence of earwax following thefirst application of the biophotonic composition up to the secondtreatment, following which there was a pronounced decrease by the timethat the third treatment was applied and thereafter an absence ofcerumen from the otitis-afflicted ear canal of the canine patients,indicating a complete cleaning of the ear canals of cerumen. FIG. 23Bpresents the earwax/cerumen measurements in all three Groups. In FIG.23B, there was an overall decrease in the presence of earwax for dogstreated with the biophotonic therapy. In both Group I (QW) and Group II(BW).

Bacterial populations were also evaluated as part of the cytologicalassessment criteria, and as can be seen from FIGS. 18A and 18B, thebacterial ecology present in the biophonic composition treated earcanals of the canine patients markedly shifted towards a greaterpresence of rod shaped bacteria (FIG. 18A) and coccoid bacteria afterthe third and fourth treatments, respectively. Yeast populations in theotitis-afflicted ear canals of the canine patients steadily decreasedafter the first treatment with the biophotonic compositions, to thepoint where by the fourth treatment; the presence of malassezia couldnot be detected (see FIG. 18C). FIGS. 24A and 24B present the bacterialecology data for all three Groups. A decrease was observed for bacterialcounts in canine patients for both the rod shaped bacteria and coccoidbacteria. FIG. 24C presents the yeast population comparison in all threeGroups.

(3) Results According to Bacteriology

Otitis externa patients were also assessed pre-treatment andpost-treatment for particular species or genera of bacteria populatingthe otitis-afflicted ear canals, and the pre-treatment results overallare presented in FIG. 19A while the post-treatment and follow-up resultsoverall are presented in FIG. 19B. Regarding the pre-treatment results,it can be seen that nearly 40% of the overall bacterial flora in theotitis-afflicted patients comprised those types of bacterial species andgenera that are associated with diseased or contaminated conditions,including Streptococcus, Pseudomonas, Escherichia coli, Shigella andCorynebacterium. For the post-treatment assessment, a shift in thebacterial flora populations was observed, with the relative amount ofStaphylococcus population and the aforementioned bacterial species andgenera greatly reduced and the overall number of colony forming unitsfrom swabs taken from the ear canals that had received the biophotoniccomposition treatment greatly reduced. FIGS. 25A-25C present thepercentages of bacterial genera in all three Groups pre-treatment andafter treatment/during follow-up.

While embodiments of the disclosure have been described above andillustrated in the accompanying figures, it will be evident to thoseskilled in the art that modifications may be made therein withoutdeparting from the essence of this disclosure. Such modifications areconsidered as possible variants comprised in the scope of thedisclosure.

INCORPORATION BY REFERENCE

All references cited in this specification, and their references, areincorporated by reference herein in their entirety where appropriate forteachings of additional or alternative details, features, and/ortechnical background.

EQUIVALENTS

While the disclosure has been particularly shown and described withreference to particular embodiments, it will be appreciated thatvariations of the above-disclosed and other features and functions, oralternatives thereof, may be desirably combined into many otherdifferent systems or applications. Also, that various presentlyunforeseen or unanticipated alternatives, modifications, variations orimprovements therein may be subsequently made by those skilled in theart which are also intended to be encompassed by the following claims.

The invention claimed is:
 1. A method of treating otitis extrena orchronic otitis extrena comprising a) applying a biophotonic compositionto a patient in need thereof, wherein the biophotonic compositioncomprises at least one oxidant and at least one chromophore capable ofactivating the oxidant and b) exposing said biophotonic composition toactinic light for a time sufficient for said chromophore to causeactivation of said oxidant.
 2. The method according to claim 1, whereinthe composition is applied to an auricle and/or ear canal of a patient.3. The method according to claim 1, wherein said biophotonic compositionis exposed to actinic light for a period of less than about 5 minutes.4. The method according to claim 1, wherein said biophotonic compositionis exposed to actinic light for a period of less than about 5 minutesper cm2 of an area to be treated.
 5. The method according to claim 1,wherein said actinic light is visible light having a wavelength betweenabout 400 nm and about 700 nm.
 6. The method according to claim 1,wherein the oxidant is chosen from hydrogen peroxide, carbamide peroxideand benzoyl peroxide.
 7. The method according to claim 1, wherein theoxidant is carbamide peroxide.
 8. The method according to claim 1,wherein the oxidant is chosen from peroxy acid and an alkali metalpercarbonate.
 9. The method according to claim 1, wherein the oxidant ispresent in an amount of from about 1% to about 10% by weight of thetotal composition.
 10. The method according to claim 1, wherein thecomposition further comprises at least one healing factor chosen fromhyaluronic acid, glucosamine and allantoin.
 11. The method according toclaim 1, wherein the composition further comprises at least one gellingagent.
 12. The method according to claim 11, wherein the gelling agentis chosen from glucose, modified starch, methyl cellulose, carboxymethylcellulose, propyl cellulose, hydroxypropyl cellulose, a carbomer,alginic acid, sodium alginate, potassium alginate, ammonium alginate,calcium alginate, agar, carrageenan, locust bean gum, pectin, andgelatin.
 13. The method according to claim 1, wherein the chromophore ischosen from a xanthene derivative dye, an azo dye, a biological stain,and a carotenoid.
 14. The method according to claim 13, wherein saidxanthene derivative dye is chosen from a fluorene dye, a fluorone dye,and a rhodole dye.
 15. The method according to claim 14, wherein saidfluorone dye is chosen from fluorescein and fluorescein derivatives. 16.The method according to claim 15, wherein said fluorescein derivative ischosen from phloxine B, rose bengal, and merbromine.
 17. The methodaccording to claim 15, wherein said fluorescein derivative is chosenfrom Eosin Y, Eosin B and Erythrosine B.
 18. The method according toclaim 17, wherein said fluorescein derivative is Eosin Y.
 19. The methodaccording to claim 1, wherein the patient is treated once a week for oneor more weeks.
 20. The method according to claim 1, wherein the patientis treated twice a week for one or more weeks.